Nucleic acids encoding anti-CD154 antibodies

ABSTRACT

The present invention relates to antagonistic antibodies specifically binding CD154, polynucleotides encoding the antibodies or fragments, and methods of making and using the foregoing.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Non-Provisional applicationSer. No. 15/228,582, filed Aug. 4, 2016, which claims the benefit ofU.S. Provisional Application Ser. No. 62/367,660, filed 28 Jul. 2016,and U.S. Provisional Application Ser. No. 62/201,150 filed 5 Aug. 2015.The entire contents of the aforementioned applications are incorporatedherein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “Sequence_Listing_065768_11470_17US5.txt”, creation date ofApr. 9, 2020, and having a size of 71 KB. The sequence listing submittedvia EFS-Web is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies specifically binding CD154,polynucleotides encoding the antibodies or fragments, and methods ofmaking and using the foregoing.

BACKGROUND OF THE INVENTION

CD154, also known as CD40 ligand (CD40L), gp39, TNF-related activationprotein (TRAP), 5c8 antigen, or T-BAM, is a trimeric transmembraneprotein of the tumor necrosis factor (TNF) superfamily. CD154 isexpressed in an activation-dependent, temporally-restricted manner onthe surface of CD4⁺ T cells. CD154 is also expressed, followingactivation, on a subset of CD8⁺ T cells, basophils, mast cells,eosinophils, natural killer cells, B cells, macrophages, dendritic cellsand platelets. CD154 also exists as a soluble form in the blood.

CD154 binds to CD40 on antigen-presenting cells (APC), which leads tovarious responses depending on the target cell type. CD40-CD154interaction is essential for normal T-B cell interactions, includingincreased co-stimulation, T-cell priming, cytokine production,antibody-class switching and affinity maturation, and antibody andautoantibody production.

Disruption of the CD40/CD154 pathway via CD154 blockage has been shownto be beneficial in autoimmune diseases such as systemic lupuserythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS),inflammatory bowel disease (IBD), type I diabetes (T1D), and allograftrejection. In humans, mutations in either CD40 or CD154 result inhyper-IgM syndrome characterized by lack of IgG or IgA isotypes (Aruffoet al., Cell 72:291, 1993).

Anti-CD154 antibodies have been described for example in Int. Pat. Publ.Nos. WO1993/08207, WO1994/10308, WO1996/40918, WO1993/009812,WO1999/051258, WO1995/006480, WO1995/006481, WO1995/006666,WO2001/002057, WO1997/017446, WO1999/012566, WO2001/068860,WO2005/003175, WO2006/033702, WO2006/030220, WO2008/118356,WO2012/052205, WO2012/138768, WO2012/138768, WO2013/055745 andWO2013/056068.

Anti-CD154 antibodies have shown to be efficacious in the treatment ofautoimmune diseases in humans. However, thromboembolism due to plateletactivation observed upon treatment prohibited continued clinicaldevelopment. Engagement of FcγRIIa on platelets has been shown to becausative for platelet activation by the anti-CD154 antibody 5c8 (Xie etal., J Immunol 192:4083-4092, 2014).

Thus, there is a need for additional anti-CD154 antibodies with improvedsafety and efficacy profiles.

SUMMARY OF THE INVENTION

The invention provides for an antagonistic antibody or an antigenbinding portion thereof specifically binding human CD154 of SEQ ID NO:1, comprising a heavy chain complementarity determining region (HCDR) 1of SEQ ID NO: 17 (SYGIS), a HCDR2 of SEQ ID NO: 23 (WISPIFGNTNYAQKFQG)and a HCDR3 of SEQ ID NO: 30 (SRYYGDLDY), wherein optionally

-   -   the HCDR1 residue S1 is mutated to A, C, D, E, G, I, K, L, M, N,        Q, R, T or V;    -   the HCDR1 residue I4 is mutated to M, L or V;    -   the HCDR1 residue S5 is mutated to A;    -   the HCDR2 residue S3 is mutated to A, T or V;    -   the HCDR2 residue P4 is mutated to V, T, L Q or E;    -   the HCDR2 residue N8 is mutated to A, C, D, E, F, G, H, I, K, L,        M, Q, R, S, T, V, W or Y;    -   the HCDR2 residue T9 is mutated to A, C, D, E, F, G, H, I, K, L,        M, Q, R, S, T, V, W or Y;    -   the HCDR2 residue N10 is mutated to A, C, D, E, F, G, H, I, K,        L, M, Q, R, S, T, V, W or Y;    -   the HCDR3 residue S1 is mutated to A or M;    -   the HCDR3 residue R2 is mutated to A, S, Q or K; and    -   the HCDR3 residue L7 is mutated to M.

The invention also provides for an isolated antagonistic antibodyspecifically binding CD154 of SEQ ID NO: 1, wherein the antibodycomprises certain VH and VL amino acid sequences as described herein.

The invention also provides for an isolated antagonistic antibodyspecifically binding CD154 of SEQ ID NO: 1, wherein the antibodycomprises certain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 amino acidsequences as described herein.

The invention also provides for an isolated antagonistic antibodyspecifically binding CD154 of SEQ ID NO: 1 comprising

-   -   the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the        LCDR3 of SEQ ID NOs: 17, 23, 30, 37, 44 and 52, respectively;    -   the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 66; or    -   the heavy chain of SEQ ID NO: 80 and the light chain of SEQ ID        NO: 81.

The invention also provides for an isolated antagonistic antibody or anantigen binding portion thereof specifically binding CD154 of SEQ ID NO:1, wherein CD154 is a homotrimer and the antibody binds a first CD154monomer in the homotrimer within amino acid residues 182-207 of CD154and a second CD154 monomer in the homotrimer within amino acid residues176-253 of CD154, wherein residue numbering is according to SEQ ID NO:1.

The invention also provides for an immunoconjugate comprising theantibody or antigen-binding portion of the antibody of the inventionlinked to a therapeutic agent or an imaging agent.

The invention also provides for a pharmaceutical composition comprisingthe antibody of the invention and a pharmaceutically accepted carrier.

The invention also provides for a polynucleotide encoding the antibodyVH of the invention, the antibody VL of the invention, or the antibodyVH and VL of the invention.

The invention also provides for a vector comprising the polynucleotideof the invention.

The invention also provides for a host cell comprising the vector of theinvention.

The invention also provides for a method of producing an antibody,comprising culturing the host cell of the invention in conditions thatthe antibody is expressed, and recovering the antibody produced by thehost cell.

The invention also provides for a method of treating an autoimmunedisease or an immune-mediated inflammatory disease comprisingadministering a therapeutically effective amount of the isolatedantibody of the invention or a pharmaceutical composition of theinvention to a patient in need thereof for a time sufficient to treatthe disease.

The invention also provides for an anti-idiotypic antibody binding tothe antibody of the invention.

The invention also provides for a kit comprising the antibody of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of antibody Fc on platelet activation byCD154:antibody immune complexes (IC). IC of soluble human CD154(shCD154, indicated as sCD40L in the Figure) and anti-CD154 antibody 5c8(IgG1 isotype) (5c8IgG1 IC) activated platelets, whereas IC of shCD154and 5c8 cloned on silenced IgG1 backbones IgG1sigma, IgG1sigma-YTE,IgG2sigma or IgG2sigma-YTE (5c8IgG1z, 5c8IgG1z-YTE, 5c8IgG2z or5c8IgG2z-YTE, respectively) had no effect. Platelet activation wasassessed as % of total platelets expressing PAC-1 (PAC-1 antibodyspecifically recognizes conformational active form of αIIbβ3 integrin)and CD62p (P-selectin surface expression). ADP: positive control. PBS:negative control. Five donors were evaluated for platelet activation.The results are shown as mean of each experiment ±SD.

FIG. 2 shows that immune complexes (IC) of shCD154 (sCD40L in theFigure) and effector silent IgG2sigma/κ anti-CD154 antibodies C4LB5,C4LB89, C4LB189, C4LB191, C4LB199 and C4LB150 do not activate platelets.Platelet activation was assessed as % of total platelets expressingPAC-1 and CD62p. IC of shCD154 and 5c8IgG1 (5c8IgG1 IC) activatedplatelets. ADP: positive control. Five donors were evaluated forplatelet activation. The results are shown as mean of eachexperiment±SD.

FIG. 3 shows that immune complexes (IC) of shCD154 (sCD40L) andanti-CD154 antibodies C4LB89 (IgG2sigma/κ), C4LB231 (IgG1sigma/κ) andC4LB232 (IgG1sigma/κ) do not activate platelets. Platelet activation wasassessed as % of total platelets expressing PAC-1 and CD62p. 5c8-IgG1 ICactivated platelets, which activation was blocked by an anti-FcγIIaantibody, indicating that platelet activation of CD154/5c8-IgG1 IC ismediated by FcγRIIa on platelets. ADP: positive control. PBS: negativecontrol. Five donors were evaluated for platelet activation. The resultsare shown as mean of each experiment±SD.

FIG. 4 shows the interaction surface between CD154 and C4LB89. Aromaticresidues F55, Y101 and Y102 in HCDR2 and HCDR3 contribute to mostinteractions (residue numbering according to SEQ ID NO: 59). Light chainof C4LB89 does not contribute to binding.

FIG. 5 shows a two-dimensional cartoon of epitope and paratope residuesidentified from crystal structure of marmoset CD154 in complex withC4LB89. The epitope residues are circled with an ellipse and theparatope residues in heavy chain HCDR1, HCDR2 and HCDR3 are shown(indicated as CDR1, CDR2 and CDR3 in the Figure). The antibody bindssimultaneously to two CD154 monomers A and B. Epitope residue numberingis according to human CD154 (SEQ ID NO: 1) and paratope residuenumbering is according to heavy chain variable region of C4LB89 (SEQ IDNO: 59).

FIG. 6A shows the alignment of residues 1-180 of human CD154 (SEQ ID NO:1, top row) and marmoset CD154 (SQ ID NO: 2, bottom row) showing thatthe C4LB89 epitope residues are conserved between human and marmosetCD1514. Epitope residues on CD154 monomer 1 are underlined, and epitoperesidues on monomer 2 are double underlined.

FIG. 6B shows the alignment of residues 181-261 of human CD154 (SEQ IDNO: 1, top row) and marmoset CD154 (SQ ID NO: 2, bottom row) showingthat the C4LB89 epitope residues are conserved between human andmarmoset CD1514. Epitope residues on CD154 monomer 1 are underlined, andepitope residues on monomer 2 are double underlined.

FIG. 7 shows that immune complexes (IC) of shCD154 (sCD40L in theFigure) and an IgG1/κ anti-CD154 antibody C4LB237 do not activateplatelets, whereas IC of shCD154 and another IgG1 antibody 5c8 activateplatelets. Antibodies alone had no effect on platelet activation, whichwas assessed as % of total platelets expressing PAC-1 and CD62p. ADP:positive control. PBS: negative control. Five donors were evaluated forplatelet activation. The results are shown as mean of eachexperiment±SD. n=4 in each group.

FIG. 8A shows that immune complexes (IC) of shCD154 (sCD40L in theFigure) and a Fc silent C4LB119 activate platelets inFcγRIIa-independent manner, whereas IC of shCD154 and antibody withC4LB119 VH/VL domains expressed on IgG1 (C4LB287) activated platelets inFcγRIIa-dependent manner. ADP: positive control. PBS: negative control.

FIG. 8B shows that immune complexes (IC) of shCD154 (sCD40L in theFigure) and a Fc silent C4LB94 activate platelets in FcγRIIa-independentmanner, whereas IC of shCD154 and antibody with C4LB94 VH/VL domainsexpressed on IgG1 (C4LB289) activated platelets in FcγRIIa-dependentmanner. ADP: positive control. PBS: negative control.

FIG. 8C shows that immune complexes (IC) of shCD154 (sCD40L in theFigure) and a Fc silent C4LB83 moderately activate platelets, whereas ICof shCD154 and antibody with C4LB83 VH/VL domains expressed on IgG1(C4LB288) activated platelets in FcγRIIa-dependent manner. ADP: positivecontrol. PBS: negative control.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patentapplications cited in this specification are herein incorporated byreference as though fully set forth.

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which the invention pertains.

As used herein and in the claims, the singular forms “a,” “and,” and“the” include plural reference unless the context clearly dictatesotherwise.

Although any methods and materials similar or equivalent to thosedescribed herein may be used in the practice for testing of the presentinvention, exemplary materials and methods are described herein. Indescribing and claiming the present invention, the following terminologywill be used.

“Specific binding” or “specifically binds” or “binds” refers to antibodybinding to an antigen or an epitope within the antigen with greateraffinity than for other antigens. Typically, the antibody binds to theantigen or the epitope within the antigen with a dissociation constant(K_(D)) of about 1×10⁻⁸ M or less, for example about 1×10⁻⁹ M or less,about 1×10⁻¹⁰ M or less, about 1×10⁻¹¹ M or less, or about 1×10⁻¹² M orless, typically with a K_(D) that is at least one hundred fold less thanits K_(D) for binding to a non-specific antigen (e.g., BSA, casein). Thedissociation constant may be measured using standard procedures.Antibodies that specifically bind to the antigen or the epitope withinthe antigen may, however, have cross-reactivity to other relatedantigens, for example to the same antigen from other species (homologs),such as human or monkey, for example Macaca fascicularis (cynomolgus,cyno), Pan troglodytes (chimpanzee, chimp) or Callithrix jacchus (commonmarmoset, marmoset). While a monospecific antibody specifically bindsone antigen or one epitope, a bispecific antibody specifically binds twodistinct antigens or two distinct epitopes.

“Neutralizing” or “neutralizes” or “neutralizing antibody” or “antibodyantagonist” or “antagonist” or “antagonistic” refers to an antibody oran antigen binding portion thereof that partially or completely inhibitsbiological activity of human CD154. Antagonistic antibodies may beidentified using assays for CD154 biological activity as describedherein. Antagonistic antibody that specifically binds human CD154 mayinhibit biological activity of human CD154 by about 20%, 30%, 40%, 50%,60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

“CD154” refers to human CD154 (hCD154) (e.g. human CD40L) protein. HumanCD154 full length protein amino acid sequence is shown in SEQ ID NO: 1.Human CD154 is found both on cell membrane as type II membrane proteinand exists as soluble form in plasma. CD154 membrane bound formcomprises residues 1-261 of SEQ ID NO: 1, with transmembrane domainpositioned between residues 23-46 and the extracellular domain spanningresidues 47-261. The soluble form of human CD154 (shCD154) is formed byproteolytic processing of the membrane bound form, and comprises theresidues 113-261 of SEQ ID NO: 1 (shCD154 amino acid sequence is shownin SEQ ID NO: 4). Both membrane bound and soluble CD154 formbiologically active trimers. “CD154” encompasses the various forms ofCD154, including monomer, dimer, trimer, membrane bound and solubleforms as well as naturally occurring variants of human CD154. Solublehuman CD154 timer (shCD154 timer) is composed of three polypeptidechains each having the amino acid sequence of SEQ ID NO: 4.

“Antibodies” as used herein is meant in a broad sense and includesimmunoglobulin molecules including monoclonal antibodies includingmurine, human, humanized and chimeric monoclonal antibodies, antibodyfragments, bispecific or multispecific antibodies, dimeric, tetramericor multimeric antibodies, single chain antibodies, domain antibodies andany other modified configuration of the immunoglobulin molecule thatcomprises an antigen binding site of the required specificity. “Fullantibody molecules” are comprised of two heavy chains (HC) and two lightchains (LC) inter-connected by disulfide bonds as well as multimersthereof (e.g. IgM). Each heavy chain is comprised of a heavy chainvariable region (VH) and a heavy chain constant region (comprised ofdomains CH1, CH2 and CH3). Each light chain is comprised of a lightchain variable region (VL) and a light chain constant region (CL). TheVH and the VL regions may be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with framework regions (FR). Each VH and VL is composed ofthree CDRs and four FR segments, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4.

“Complementarity determining regions (CDR)” are “antigen binding sites”in an antibody. CDRs may be defined using various terms: (i)Complementarity Determining Regions (CDRs), three in the VH (HCDR1,HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based onsequence variability (Wu and Kabat, J Exp Med 132:211-50, 1970; Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991).(ii) “Hypervariable regions”, “HVR”, or “HV”, three in the VH (H1, H2,H3) and three in the VL (L1, L2, L3) refer to the regions of antibodyvariable domains which are hypervariable in structure as defined byChothia and Lesk (Chothia and Lesk, Mol Biol 196:901-17, 1987). TheInternational ImMunoGeneTics (IMGT) database (www_imgt_org) provides astandardized numbering and definition of antigen-binding sites. Thecorrespondence between CDRs, HVs and IMGT delineations is described inLefranc et al., Dev Comparat Immunol 27:55-77, 2003. The terms “CDR”,“HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2” and “LCDR3” includes CDRsdefined by any of the methods described supra, Kabat, Chothia or IMGT,unless otherwise explicitly stated in the specification.

Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE,IgG and IgM, depending on the heavy chain constant domain amino acidsequence. IgA and IgG are further sub-classified as the isotypes IgA₁,IgA₂, IgG₁, IgG₂, IgG₃ and IgG₄. Antibody light chains of any vertebratespecies can be assigned to one of two clearly distinct types, namelykappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

“Antibody fragments” or “antigen binding portion of an antibody” refersto a portion of an immunoglobulin molecule that retains the heavy chainand/or the light chain antigen binding site, such as heavy chaincomplementarity determining regions (HCDR) 1, 2 and 3, light chaincomplementarity determining regions (LCDR) 1, 2 and 3, a heavy chainvariable region (VH), or a light chain variable region (VL). Antibodyfragments include well known Fab, F(ab′)2, Fd and Fv fragments as wellas domain antibodies (dAb) consisting one VH domain. VH and VL domainsmay be linked together via a synthetic linker to form various types ofsingle chain antibody designs where the VH/VL domains pairintramolecularly, or intermolecularly in those cases when the VH and VLdomains are expressed by separate single chain antibody constructs, toform a monovalent antigen binding site, such as single chain Fv (scFv)or diabody; described for example in Int. Pat. Publ. No. WO1998/44001,Int. Pat. Publ. No. WO1988/01649; Int. Pat. Publ. No. WO1994/13804; Int.Pat. Publ. No. WO1992/01047.

“Monoclonal antibody” refers to an antibody population with single aminoacid composition in each heavy and each light chain, except for possiblewell known alterations such as removal of C-terminal lysine from theantibody heavy chain. Monoclonal antibodies typically bind one antigenicepitope, except that bispecific monoclonal antibodies bind two distinctantigenic epitopes. Monoclonal antibodies may have heterogeneousglycosylation within the antibody population. Monoclonal antibody may bemonospecific or multispecific, or monovalent, bivalent or multivalent. Abispecific antibody is included in the term monoclonal antibody.

“Isolated antibody” refers to an antibody or antibody fragment that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody specifically binding CD154 issubstantially free of antibodies that specifically bind antigens otherthan CD154. “Isolated antibody” encompasses antibodies that are isolatedto a higher purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% pure.

“Chothia residues” are the antibody VL and VH residues numberedaccording to Al-Lazikani (Al-Lazikani et al., J Mol Biol 273:927-48,1997).

“Humanized antibodies” refers to antibodies in which the antigen bindingsites are derived from non-human species and the variable regionframeworks are derived from human immunoglobulin sequences. Humanizedantibodies may include substitutions in the framework regions so thatthe framework may not be an exact copy of expressed human immunoglobulinor germline gene sequences.

“Human antibodies” refers to antibodies having heavy and light chainvariable regions in which both the framework and the antigen bindingsite are derived from sequences of human origin. If the antibodycontains a constant region or a portion of the constant region, theconstant region also is derived from sequences of human origin.

Human antibody comprises heavy or light chain variable regions that are“derived from” sequences of human origin if the variable regions of theantibody are obtained from a system that uses human germlineimmunoglobulin or rearranged immunoglobulin genes. Such exemplarysystems are human immunoglobulin gene libraries displayed on phage, andtransgenic non-human animals such as mice or rats carrying humanimmunoglobulin loci as described herein. “Human antibody” may containamino acid differences when compared to the human germline or rearrangedimmunoglobulin sequences due to for example naturally occurring somaticmutations or intentional introduction of substitutions into theframework or antigen binding site, or both. Typically, “human antibody”is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in aminoacid sequence to an amino acid sequence encoded by a human germline orrearranged immunoglobulin gene. In some cases, “human antibody” maycontain consensus framework sequences derived from human frameworksequence analyses, for example as described in Knappik et al., J MolBiol 296:57-86, 2000, or synthetic HCDR3 incorporated into humanimmunoglobulin gene libraries displayed on phage, for example asdescribed in Shi et al., J Mol Biol 397:385-96, 2010 and Int. Pat. Publ.No. WO2009/085462.

Isolated humanized antibodies are synthetic. Human antibodies, whilederived from human immunoglobulin sequences, may be generated usingsystems such as phage display incorporating synthetic CDRs and/orsynthetic frameworks, or may be subjected to in vitro mutagenesis toimprove antibody properties, resulting in antibodies that are notexpressed by the human antibody germline repertoire in vivo.

Antibodies in which antigen binding sites are derived from a non-humanspecies are not included in the definition of “human antibody”.

“Recombinant” includes antibodies and other proteins that are prepared,expressed, created or isolated by recombinant means.

“Epitope” as used herein refers to a portion of an antigen to which anantibody specifically binds. Epitopes usually consist of chemicallyactive (such as polar, non-polar or hydrophobic) surface groupings ofmoieties such as amino acids or polysaccharide side chains and may havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. An epitope may be composed ofcontiguous and/or discontiguous amino acids that form a conformationalspatial unit. For a discontiguous epitope, amino acids from differingportions of the linear sequence of the antigen come in close proximityin 3-dimensional space through the folding of the protein molecule.

“Paratope” refers to a portion of an antibody to which an antigenspecifically binds. A paratope may be linear in nature or may bediscontinuous, formed by a spatial relationship between non-contiguousamino acids of an antibody rather than a linear series of amino acids. A“light chain paratope” and a “heavy chain paratope” or “light chainparatope amino acid residues” and “heavy chain paratope amino acidresidues” refer to antibody light chain and heavy chain residues incontact with an antigen, respectively, or in general, “antibody paratoperesidues” refer to those antibody amino acids that are in contact withantigen.

“Bispecific” refers to an antibody that specifically binds two distinctantigens or two distinct epitopes within the same antigen. Thebispecific antibody may have cross-reactivity to other related antigensor can bind an epitope that is shared between two or more distinctantigens.

“Multispecific” refers to an antibody that specifically binds at leasttwo distinct antigen or at least two distinct epitopes within the sameantigen. Multispecific antibody may bind for example two, three, four orfive distinct antigens or distinct epitopes within the same antigen.

“In combination with” means that the drugs or therapeutics areadministered to a subject such as human together in a mixture,concurrently as single agents or sequentially as single agents in anyorder.

“CD154 biological activity” refers to any activity occurring as a resultof CD154 binding to its receptor CD40. CD154 biological activity may befor example CD154-mediated activation of CD40⁺ B cells or dendriticcells (DC), or downstream activation of CD40 signaling pathways. CD154biological activity may be measured using well known methods and methodsdescribed herein, such as measuring CD154-mediated B cell proliferationor B cell activation by assessing ICAM-1 up-regulation or increasedcytokine production by the B cells, CD154-mediated DC activation byassessing increased surface expression of CD80 and/or CD86 or cytokinesecretion by the DC cells, or activation of CD40 signaling pathway asassessed by reporter gene assays such as measuring secretion of secretedembryonic alkaline phosphatase (SEAP) by cells expressing SEAP under thecontrol of NF-κB-inducible promoter.

“Vector” means a polynucleotide capable of being duplicated within abiological system or that can be moved between such systems. Vectorpolynucleotides typically contain elements, such as origins ofreplication, polyadenylation signal or selection markers, that functionto facilitate the duplication or maintenance of these polynucleotides ina biological system. Examples of such biological systems may include acell, virus, animal, plant, and reconstituted biological systemsutilizing biological components capable of duplicating a vector. Thepolynucleotide comprising a vector may be DNA or RNA molecules or ahybrid of these.

“Expression vector” means a vector that can be utilized in a biologicalsystem or in a reconstituted biological system to direct the translationof a polypeptide encoded by a polynucleotide sequence present in theexpression vector.

“Polynucleotide” means a molecule comprising a chain of nucleotidescovalently linked by a sugar-phosphate backbone or other equivalentcovalent chemistry. Double and single-stranded DNA and RNA are typicalexamples of polynucleotides.

“Polypeptide” or “protein” means a molecule that comprises at least twoamino acid residues linked by a peptide bond to form a polypeptide.Small polypeptides of less than 50 amino acids may be referred to as“peptides”.

Conventional one and three-letter amino acid codes are used herein asshown in Table 1.

TABLE 1 Three-letter One-letter Amino acid code code Alanine Ala AArginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys C GlutamateGln E Glutamine Glu Q Glycine Gly G Histidine His H Isoleucine Ile ILysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P SerineSer S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val VCompositions of Matter

The present invention provides antagonistic antibodies specificallybinding CD154 with high affinity and efficiently neutralizing CD154biological activity. The invention is based, at least in part, on theidentification that contrary to the current understanding that bindingof the antibodies specifically binding CD154 to the FcγRIIa on plateletsresults in activation and aggregation of platelets and subsequentthromboembolism, it has been herein discovered that platelet activationalso depends on the CD154 epitope the antibodies binds to. It has beendiscovered herein that antibodies of the invention binding certainepitope on CD154 which are capable of engaging FcγRIIa do not to mediateplatelet activation. In addition, the antibodies of the invention areoptionally Fc engineered to prevent triggering of additional unwantedimmunostimulatory functions. Therefore, the antibodies of the inventionmay have more favorable safety profiles in the clinical setting whencompared to existing antibodies specifically binding CD154.

CD154 is a target in autoimmunity, graft rejection and otherimmune-related diseases in mice, non-human primates (NP) and humans. Inseveral Phase II Clinical Trials, antibodies specifically binding CD154have been shown to effectively block the activities of CD154 in vivo andameliorate disease. CD154 antagonists are distinct from all othertherapeutics in its impact on the immune response; they are the onlytherapeutics that can induce functional immunological tolerance, asdemonstrated both in mice and monkeys. In mice, virtually all autoimmunedisease models can be effectively ameliorated with CD154 antagonists(Noelle et al., Ann N Y Acad Sci 815: 384-391, 1997; Mackey et al., JLeukoc Biol 63: 418-428, 1998; Noelle, Agents Actions Suppl 49: 17-22,1998; Quezada et al., Annu Rev Immunol 22: 307-328, 2004), withlong-term remission observed.

The invention provides for an isolated antagonistic antibody or anantigen binding portion thereof specifically binding CD154 of SEQ ID NO:1.

The invention also provides for an isolated antagonistic antibody or anantigen binding portion thereof specifically binding CD154, whereinCD154 is a homotrimer and the antibody binds a first CD154 monomer inthe homotrimer within amino acid residues 182-207 of CD154 and a secondCD154 monomer in the homotrimer within amino acid residues 176-253 ofCD154, wherein residue numbering is according to SEQ ID NO: 1.

Such exemplary antibody is the antibody C4LB89. Since antibody C4LB235and C4LB236 variable regions differ by one amino acid residue in theLCDR2 when compared to those of C4LB89, and since C4LB231 and C4LB232have identical VH/VL sequences with C4LB89, it is expected that alsothese antibodies bind the same CD154 epitope as C4LB89. Antibodies thatbind CD154 within residues 182-207 and 176-253 are incapable ofactivating platelets even when capable of engaging with FcγR, includingFcγRIIa. Therefore, these antibodies may have improved safety profilewhen compared to other antagonistic antibodies specifically bindingCD154.

The invention also provides for an antagonistic antibody or an antigenbinding portion thereof specifically binding human CD154 of SEQ ID NO:1, comprising a heavy chain complementarity determining region (HCDR) 1of SEQ ID NO: 17 (SYGIS), a HCDR2 of SEQ ID NO: 23 (WISPIFGNTNYAQKFQG)and a HCDR3 of SEQ ID NO: 30 (SRYYGDLDY), wherein optionally

-   -   the HCDR1 residue S1 is mutated to A, C, D, E, G, I, K, L, M, N,        Q, R, T or V;    -   the HCDR1 residue I4 is mutated to M, L or V;    -   the HCDR1 residue S5 is mutated to A;    -   the HCDR2 residue S3 is mutated to A, T or V;    -   the HCDR2 residue P4 is mutated to V, T, L Q or E;    -   the HCDR2 residue N8 is mutated to A, C, D, E, F, G, H, I, K, L,        M, Q, R, S, T, V, W or Y;    -   the HCDR2 residue T9 is mutated to A, C, D, E, F, G, H, I, K, L,        M, Q, R, S, T, V, W or Y;    -   the HCDR2 residue N10 is mutated to A, C, D, E, F, G, H, I, K,        L, M, Q, R, S, T, V, W or Y;    -   the HCDR3 residue S1 is mutated to A or M;    -   the HCDR3 residue R2 is mutated to A, S, Q or K; and    -   the HCDR3 residue L7 is mutated to M.

In some embodiments, the antibody of the invention comprises a lightchain complementarity determining region (LCDR) 1 of SEQ ID NO: 37(RASQSISSYLN), a LCDR2 of SEQ ID NO: 44 (YANSLQS) and a LCDR3 of SEQ IDNO: 52 (QQSDSIPWT), wherein optionally

-   -   the LCDR1 residue Q4 is mutated to A, C, D, E, F, G, H, I, K, L,        M, N, R, S, T, V, W or Y;    -   the LCDR1 residue S5 is mutated to A, C, D, E, F, G, H, I, K, L,        M, N, Q, R, T, V, W or Y;    -   the LCDR1 residue S7 is mutated to A, C, D, E, F, G, H, I, K, L,        M, N, Q, R, T, V, W or Y;    -   the LCDR1 residue S8 is mutated to A, C, D, E, F, G, H, I, K, L,        M, N, Q, R, T, V, W or Y;    -   the LCDR2 residue A2 is mutated to S;    -   the LCDR2 residue N3 is mutated to A, C, D, E, F, G, H, I, K, L,        M, Q, R, S, T, V, W or Y;    -   the LCDR2 residue S4 is mutated to A, C, D, E, F, G, H, I, K, L,        M, N, Q, R, T, V, W or Y;    -   the LCDR2 residue L5 is mutated to A, C, D, E, F, G, H, I, K, M,        N, Q, R, S, T, V, W or Y;    -   the LCDR2 residue Q6 is mutated to E, D or N;    -   the LCDR2 residue S7 is mutated to A, C, D, E, F, G, H, I, K, L,        M, N, Q, R, T, V, W or Y;    -   the LCDR3 residue S3 is mutated to A;    -   the LCDR3 residue D4 is mutated to N;    -   the LCDR3 residue S5 is mutated to A, C, D, E, F, G, H, I, K, L,        M, N, Q, R, T, V, W or Y; and    -   the LCDR3 residue 6 is mutated to A, C, D, E, G, K, L, M, N, Q,        R, S, T or V.

A crystal structure of a complex of antibody C4LB89 and CD154 revealedthat the antibody binds CD154 with VH residues only. Further analysesindicated that certain substitutions as shown in Table 19 and Table 20as indicated supra, in the CDRs of the antibody are not expected toaffect the overall structure of the complex and thereforecharacteristics of the antibody.

In some embodiments, the antibody of the invention comprises a lightchain complementarity determining region (LCDR) 1, a LCDR2 and a LCDR3of

SEQ ID NOs: 36, 43 and 51, respectively;

SEQ ID NOs: 37, 44 and 52, respectively;

SEQ ID NOs: 38, 45 and 53, respectively;

SEQ ID NOs: 39, 46 and 54, respectively;

SEQ ID NOs: 40, 47 and 55, respectively;

SEQ ID NOs: 41, 47 and 56, respectively;

SEQ ID NOs: 42, 48 and 57, respectively;

SEQ ID NOs: 37, 49 and 52, respectively; or

SEQ ID NOs: 37, 50 and 52, respectively.

In some embodiments, the antibody of the invention comprises the LCDR1,the LCDR2 and the LCDR3 of SEQ ID NOs: 37, 44 and 52, respectively.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:17, 23, 30, 37, 44 and 52, respectively.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:17, 23, 30, 37, 49 and 52, respectively.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs:17, 23, 30, 37, 50 and 52, respectively.

In some embodiments, an immune complex of the antibody of the inventionand soluble human CD154 (shCD154) does not activate platelets, whereinplatelet activation is measured by P-selectin surface expression onplatelets.

Platelet activation is a well-known process that converts the smooth,nonadherent platelet into a sticky spiculated particle that releases andexpresses biologically active substances and acquires the ability tobind the plasma protein fibrinogen. Activation may also occur as aresult of the physical stimulus of high fluid shear stress, such as thatfound at the site of a critical arterial narrowing (Quinn et al., 2005,Platelet Function: assessment, diagnosis, and treatment, Humana Press,pp. 3-20). Activation of platelets results in activation ofintracellular signaling pathways resulting in upregulation of plateletsurface expression of P-selectin and increased binding affinity offibrinogen to integrin receptors αIIbβ3. Platelet activation maytherefore be measured by measuring increased P-selectin surfaceexpression or binding of probe ligand e.g. PAC-1 to αIIbβ3 integrin onplatelets using for example flow cytometry. The antibodies of theinvention do not activate human platelets when the antibody in complexwith shCD154 does not elevate surface expression of P-selectin orincrease binding of probe ligand (e.g. PAC-1) to αIIbβ3 integrin instatistically significant manner when compared to the surface expressionof P-selectin and increased binding of probe ligand (e.g. PAC-1) toαIIbβ3 integrin induced by shCD154.

In some embodiments, the antibody of the invention has at least one ofthe following properties:

-   -   binds to CD154 with a dissociation constant (K_(D)) of about        5×10⁻⁹ M or less, when the K_(D) is measured using ProteOn™        XPR36 system at 25° C. in Dulbecco's phosphate buffered saline        containing 0.03% polysorbate P20 and 100 μg/ml bovine serum        albumin; inhibits CD154-mediated human B cell proliferation with        an IC₅₀ value of about 2.7×10⁻⁹ M or less; or

inhibits CD154-mediated expression of secreted embryonic alkalinephosphatase (SEAP) under NF-κB-inducible interferon-β (IFN-β) minimalpromoter in HEK293 cells stably expressing SEAP and human CD40 with anIC₅₀ value of about 2.1×10⁻⁸ M or less.

In some embodiments, the antibody of the invention binds CD154 with adissociation constant (K_(D)) of about 5×10⁻⁹ M or less, about 1×10⁻⁹ Mor less, about 5×10⁻¹⁰ M or less, about 1×10⁻¹⁰ M or less, about 5×10⁻¹¹M or less, or about 1×10⁻¹¹ M or less.

In some embodiments, the antibody specifically binding CD154cross-reacts with Macaca fascicularis (cyno) CD154 or Callithrix jacchus(marmoset) CD154.

The affinity of an antibody to human, cyno or marmoset CD154 may bedetermined experimentally using any suitable method. Such methods mayutilize ProteOn™ XPR36, Biacore 3000 or KinExA® instrumentation, ELISAor competitive binding assays known to those skilled in the art. Themeasured affinity of a particular antibody to CD154 may vary if measuredunder different conditions (e.g., osmolarity, pH). Thus, measurements ofaffinity and other binding parameters (e.g., K_(D), K_(on), K_(off)) aretypically made with standardized conditions and a standardized buffer,such as the buffer described herein. Skilled in the art will appreciatethat the internal error for affinity measurements for example usingBiacore 3000 or ProteOn™ (measured as standard deviation, SD) cantypically be within 5-33% for measurements within the typical limits ofdetection. Therefore the term “about” reflects the typical standarddeviation in the assay. For example, the typical SD for a K_(D) of1×10⁻⁹ M is up to ±0.33×10⁻⁹ M.

In some embodiments, the antibody of the invention inhibitsCD154-mediated human B cell proliferation with an IC₅₀ value of about2.7×10⁻⁹ M or less.

In the B cell proliferation assay, 1×10⁵ human tonsil B cells may becultured with 100 ng/ml recombinant human IL-21, 0.5 μg/ml trimericrecombinant soluble human CD154 expressed as a leucine zipper fusionprotein and anti-CD154 antibodies in a range of 0.000064-25 μg/ml in afinal volume of 200 μl/well. After 2 days incubation methyl(˜3H)-Thymidine (0.5 μCi/well) may be added to the cultures and effectof the antibodies on human B cell proliferation may be determined afterovernight incubation.

In some embodiments, the antibody of the invention inhibitsCD154-induced expression of secreted embryonic alkaline phosphatase(SEAP) under NF-κB-inducible interferon-β (IFN-β) minimal promoter inHEK293 cells stably expressing SEAP and human CD40 with an IC₅₀ value ofabout 2.1×10⁻⁸ M or less.

In some embodiments, the antibody of the invention inhibitsCD154-induced expression of secreted embryonic alkaline phosphatase(SEAP) under NF-κB-inducible IFN-β minimal promoter in HEK293 cellsstably expressing SEAP and human CD40 with an IC₅₀ value of betweenabout 2.1×10⁻⁸ M and 5.4×10⁻¹⁰ M.

The cells that may be used are for example HEK-Bue™ CD40L cells(InvivoGen, San Diego, Calif.). Human CD154 may be provided as trimericsoluble CD154-leucine zipper fusion protein. Signal from the secretedalkaline phosphatase may be detected and an IC₅₀ may be calculated forthe inhibition using well known methods.

In some embodiments, the antibody of the invention binds a first CD154monomer and a second CD154 monomer simultaneously in a CD154 homotrimer.

In some embodiments, the antibody of the invention binds at least one,two, three, four, five, six, seven or eight CD154 residues in the firstCD154 monomer within amino acid residues 182-207 of CD154 of SEQ ID NO:1.

In some embodiments, the antibody of the invention binds at least one,two, three, four, five, six, seven or eight CD154 residues in the secondCD154 monomer within amino acid residues 176-253 of CD154 of SEQ ID NO:1.

In some embodiments, the antibody of the invention binds residues E182,S185, Q186, A187, P188, S214, A215 and R207 in the first CD154 monomer,wherein residue numbering is according to SEQ ID NO: 1.

In some embodiments, the antibody of the invention binds residues T176,F177, C178, Q220, S248, H249, G250 and F253 in the second CD154 monomer,wherein residue numbering is according to SEQ ID NO: 1.

“Within” means that the antibody binds only residues inside the aminoacid stretches 182-207, 176-354 or 182-207 and 176-354.

Such exemplary antibody is the antibody C4LB89. Since antibody C4LB235and C4LB236 variable regions differ by one amino acid residue in theLCDR2 when compared to those of C4LB89, and since C4LB231 and C4LB232have identical VH/VL sequences with C4LB89, it is expected that alsothese antibodies bind the same CD154 epitope as C4LB89.

In some embodiments, the antibody of the invention binds human CD154with paratope residues that reside in the VH of the antibody.

“Paratope residue” is a residue in the antibody VH or VL that resideswithin 4 Å from the CD154 residues. Paratope residues may be identifiedfrom crystal structures of the complex of antibody with CD154.

An exemplary antibody that binds CD154 with VH paratope residues onlywithout VL in contact with antigen is an antibody that comprises the VHand the VL of antibody C4LB89. Since C4LB235 and C4LB236 variableregions differ by one amino acid residue in the LCDR2 when compared tothose of C4LB89, and since C4LB231 and C4LB232 have identical VH/VLsequences with C4LB89, it is expected that also these antibodies bindCD154 with VH residues only. Antibodies that bind CD154 within residues182-207 or 176-354 of SEQ ID NO: 1 or residues E182, S185, Q186, A187,P188, S214, A215 and R207 in the first CD154 monomer and residues T176,F177, C178, Q220, S248, H249, G250 and F253 in the second CD154 monomerin the CD154 homotrimer are incapable of activating platelets even whencapable of engaging with FcγR, including FcγRIIa. Therefore, theseantibodies may have an improved safety provide when compared to otherantibodies that specifically bind CD154.

In some embodiments, the antibody the invention comprises a heavy chainvariable region (VH) of SEQ ID NO: 59, optionally the VH comprising one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen or fifteen amino acid substitutions.

In some embodiments, the antibody of the invention comprises the heavychain variable region (VH) of SEQ ID NO: 59.

The antibody comprising the HCDR1, the HCDR2 and the HCDR3 of SEQ IDNOs: 17, 23 and 30, respectively, or having the HCDRs of VH of SEQ IDNO: 59 or the VH of SEQ ID NO: 59 binds CD154 with the antibody VH only.Therefore, the VH of SEQ ID NO: 59 or the VH comprising the HCDR1, theHCDR2 and the HCDR3 of SEQ ID NOs: 17, 23 and 30, respectively, may becombined with any light chain variable region (VL) sequence and thebinding of the resulting antibody to CD154 may be tested using assaysdescribed herein to generate an antibody that specifically binds CD154.

For example, the VH comprising the HCDR1, the HCDR2 and the HCDR3 of SEQID NOs: 17, 23 and 30, respectively, or the VH of SEQ ID NO: 59 may beused to screen for VL domains capable of forming a two-domain specificantigen-binding fragment capable of binding to CD154. The screening maybe accomplished by phage display screening methods using for examplehierarchical dual combinatorial approach disclosed in PCT Publ. No.WO1992/01047. In this approach, an individual colony containing either aH or L chain clone, for example VH of SEQ ID NO: 59, is used to infect acomplete library of clones encoding the other chain (L or H), and theresulting two-chain specific antigen-binding domain is selected inaccordance with phage display techniques as described herein and testedfor its binding and antagonistic activity towards CD154.

Alternatively, the VH or SEQ ID NO: 59 or the VH comprising the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 17, 23 and 30, respectively, maybe combined with VL domains of existing CD154 antibodies or CD154antibodies described herein, and the resulting antibody is tested forits binding and antagonistic activity towards CD154.

In some embodiments, the antibody of the invention comprises a lightchain variable region (VL) of SEQ ID NOs: 65, 66, 67, 68, 69, 70, 71, 72or 73, optionally the VL comprising one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteenamino acid substitutions.

In some embodiments, the antibody of the invention comprises the VH ofSEQ ID NO: 59 and the VL or SEQ ID NOs: 65, 66, 67, 68, 69, 70, 71, 72or 73.

In some embodiments, the antibody of the invention comprises a lightchain variable region (VL) of SEQ ID NOs: 66, 72 or 73, optionally theVL comprising one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions.

In some embodiments, the antibody of the invention comprises the VH ofSEQ ID NO: 59 and the VL of SEQ ID NO: 66.

In some embodiments, the antibody of the invention comprises the VH ofSEQ ID NO: 59 and the VL of SEQ ID NO: 72.

In some embodiments, the antibody of the invention comprises the VH ofSEQ ID NO: 59 and the VL of SEQ ID NO: 73.

In some embodiments, the antibody of the invention comprising at leastone substitution in an Fc region, wherein the antibody does not activatehuman platelets.

In some embodiments, the antibody of the invention has reduced bindingto FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa or FcγRIIIb.

“Reduced binding” refers to reduced binding of the antibodies of theinvention having at least one substitution in the Fc region to an FcγRreceptor when compared to the binding of the parental antibody withoutthe substitution to the same FcγR receptor. “Reduced binding” may be atleast about 100-fold, at least about 500-fold, at least about 1000-fold,at least about 5000-fold, at least about 10,000-fold, or at least about20,000-fold reduced binding. In practice, antibodies exhibiting “reducedbinding” to a particular FcγR refer to antibodies that havestatistically insignificant effector function mediated by the particularFcγR.

In some embodiments, the antibody of the invention comprises at leastone substitution in the Fc region.

In some embodiments, the at least one substitution in the Fc region is asubstitution L234A, L235A, G237A, P238S, M252Y, S254T, T256E, H268A,A330S or P331S, wherein residue numbering is according to the EU Index.

In some embodiments, the antibody of the invention comprisessubstitutions L234A, L235A, G237A, P238S, H268A, A330S or P331S in theFc region, wherein residue numbering is according to the EU Index.

In some embodiments, the at least one substitution in the Fc region is asubstitution V234A, G237A, P238S, M252Y, S254T, T256E H268A, V309L,A330S or P331S, wherein residue numbering is according to the EU Index.

In some embodiments, the antibody of the invention comprisessubstitutions V234A, G237A, P238S, H268A, V309L, A330S and P331S in theFc region, wherein residue numbering is according to the EU Index.

The invention also provides for an antagonistic antibody or an antigenbinding portion thereof specifically binding CD154 of SEQ ID NO: 1,comprising a heavy chain of SEQ ID NO: 80 and a light chain of SEQ IDNO: 81.

The invention also provides for an antagonistic antibody or an antigenbinding portion thereof specifically binding CD154 of SEQ ID NO: 1,comprising a heavy chain of SEQ ID NO: 82 and a light chain of SEQ IDNO: 81.

The invention also provides for an antagonistic antibody or an antigenbinding portion thereof specifically binding CD154 of SEQ ID NO: 1,comprising a heavy chain of SEQ ID NO: 83 and a light chain of SEQ IDNO: 81.

(C4LB89 VH on IgG1sigma: C4LB231 HC) SEQ ID NO: 80QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGWISPIFGNTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSRYYGDLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Light chain of C4LB89 and C4LB231) SEQ ID NO: 81DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYANSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSDSIPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC(C4LB89 VH on IgG1sigmaYTE) SEQ ID NO: 82QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGWISPIFGNTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSRYYGDLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGASSVFLFPPKPKDTLYITREPEVTCVVVDVSAEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(C4LB89 VH on IgG2sigma) SEQ ID NO: 83QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGISWVRQAPGQGLEWMGWISPIFGNTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSRYYGDLDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPAAASSVFLFPPKPKDTLMISRTPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Immune effector properties of the antibodies of the invention may beenhanced or silenced through Fc modifications by techniques known tothose skilled in the art. For example, Fc effector functions such as C1qbinding, complement dependent cytotoxicity (CDC), antibody-dependentcell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cellsurface receptors (e.g., B cell receptor; BCR), etc. may be providedand/or controlled by modifying residues in the Fc responsible for theseactivities. For example, Fc substitutions V234A/G237A/P238S,V234A/G237A/H268Q, H268A/V309L/A330S/P331 orV234A/G237A/P238S/H268A/V309L/A330S/P331S (Intl. Pat. Publ. No.WO11/066501) or L234A/L235A/G237A/P238S/H268A/A330S/P331S may beintroduced to the antibodies of the invention.

Binding of the antibodies of the invention to FcγRI, FcγRIIa, FcγRIIb,FcγRIIIa and FcγRIIIb may be evaluated using recombinant soluble formsor cell-associated forms of the Fcγ receptors. For example, direct orindirect, e.g., competitive binding, measurements may be applied forassessing relative affinities and avidities of the antibodies of theinvention to various FcγR. In an exemplary assay, test antibody bindingto soluble FcγR captured on a plate is evaluated using competitivebinding between 1 μg/ml biotinylated human IgG1 and serial dilutions oftest antibody pre-complexed with antigen.

In some embodiments, the antibody of the invention has reduced antibodydependent cellular cytotoxicity (ADCC), antibody-dependent cellularphagocytosis” (“ADCP”) and/or complement dependent cytotoxicity (CDC).

“Antibody-dependent cellular cytotoxicity”, “antibody-dependentcell-mediated cytotoxicity” or “ADCC” is a mechanism for inducing celldeath that depends upon the interaction of antibody-coated target cellswith effector cells possessing lytic activity, such as natural killercells, monocytes, macrophages and neutrophils via Fc gamma receptors(FcγR) expressed on effector cells. For example, NK cells expressFcγRIIIa, whereas monocytes express FcγRI, FcγRII and FcγRIIIa. Toassess ADCC activity of the antibodies of the invention, the antibodymay be added to target cells in combination with immune effector cells,which may be activated by the antigen antibody complexes resulting incytolysis of the target cell. Cytolysis is generally detected by therelease of label (e.g. radioactive substrates, fluorescent dyes ornatural intracellular proteins) from the lysed cells. Exemplary effectorcells for such assays include peripheral blood mononuclear cells (PBMC)and NK cells. Exemplary target cells include D1.1 Jurkat cells (ATCC®CRL-10915™) or T cells expressing CD154.

“Antibody-dependent cellular phagocytosis” (“ADCP”) refers to amechanism of elimination of antibody-coated target cells byinternalization by phagocytic cells, such as macrophages or dendriticcells. ADCP may be evaluated using monocyte-derived macrophages aseffector cells and D1.1 Jurkat cells expressing CD154 engineered toexpress GFP or other labeled molecule as target cells. Effctor:targetcell ratio may be for example 4:1. Effector cells may be incubated withtarget cells for 4 hours with or without the test CD154 antibody. Afterincubation, cells may be detached using accutase. Macrophages may beidentified with anti-CD11b and anti-CD14 antibodies coupled to afluorescent label, and percent phagocytosis may be determined based on %GFP fluorescent in the CD11⁺CD14⁺ macrophages using standard methods.

“Complement-dependent cytotoxicity”, or “CDC”, refers to a mechanism forinducing cell death in which an Fc effector domain of a target-boundantibody binds and activates complement component C1q which in turnactivates the complement cascade leading to target cell death.Activation of complement may also result in deposition of complementcomponents on the target cell surface that facilitate ADCC by bindingcomplement receptors (e.g., CR3) on leukocytes. CDC of CD154-expressingcells may be measured for example by plating Jurkat cells in anappropriate medium, adding anti-CD154 antibodies into the mixture,followed by addition of pooled human serum. After incubation period,percentage (%) lysed cells may be detected as % propidium iodide stainedcells in FACS assay using standard methods.

“Reduced ADCC”, “reduced CDC” and “reduced ADCP” refers toantibody-induced ADCC, CDC and/or ADCP that is statisticallyinsignificant in standard assays that measure ADCC, CDC and/or ADCP,such as assays described herein and in assays described in U.S. Pat. No.8,871,204.

The antibodies of the invention with a desired affinity andneutralization profile may be selected from libraries of variants orfragments by panning with human CD154 or marmoset CD154 and optionallyby further antibody affinity maturation. In an exemplary panningcampaign, phage libraries may be panned with marmoset CD154.Alternatively, antibodies of the invention may be generated byimmunizing mice with human CD154 or marmoset CD154 or both, andscreening the hybriomas for binding to human CD154, and subsequentlyassessing the antagonistic properties of the antibodies using methodsdescribed herein.

In some embodiments, the antibody of the invention competes for bindingto CD154 with an antibody comprising the VH of SEQ ID NO: 59 and the VLof SEQ ID NO: 66.

Competition between specific binding to CD154 with antibodies of theinvention comprising certain VH and VL sequences may be assayed in vitrousing well known methods. For example, binding of MSD Sulfo-Tag™NHS-ester-labeled antibody to CD154 in the presence of an unlabeledantibody may be assessed by ELISA, or Bioacore analyses or flowcytometry may be used to demonstrate competition with the antibodies ofthe current invention. The antibody competes for binding to CD154 with areference antibody (e.g. an antibody comprising the VH of SEQ ID NO: 59and the VL of SEQ ID NO: 66) when the antibody inhibits binding of thereference antibody to CD154 by 80% or more, for example 85% or more, 90%or more, or 95% or more.

In some embodiments, the VH of SEQ ID NO: 59 may be combined with the VLof any of the anti-CD154 antibodies described in Int. Pat. Publ. Nos.WO1993/08207, WO1994/10308, WO1996/40918, WO1993/009812, WO1999/051258,WO1995/006480, WO1995/006481, WO1995/006666, WO2001/002057,WO1997/017446, WO1999/012566, WO2001/068860, WO2005/003175,WO2006/033702, WO2006/030220, WO2008/118356, WO2012/052205,WO2012/138768, WO2012/138768, WO2013/055745 and WO2013/056068 togenerate an antagonistic anti-CD154 antibody. The binding andantagonistic activity of the resulting antibodies may be tested usingassays and protocols described herein.

The invention also provides for an antagonistic antibody specificallybinding a CD154 of SEQ ID NO: 1, comprising the HCDR1, the HCDR2 and theHCDR3 of

-   -   SEQ ID NOs: 16, 22 and 29, respectively;    -   SEQ ID NOs: 17, 23 and 30, respectively;    -   SEQ ID NOs: 16, 24 and 31, respectively;    -   SEQ ID NOs: 18, 25 and 32, respectively;    -   SEQ ID NOs: 19, 26 and 33, respectively;    -   SEQ ID NOs: 20, 27 and 34, respectively; or    -   SEQ ID NOs: 21, 28 and 35, respectively.

The invention also provides for an antagonistic antibody specificallybinding CD154 of SEQ ID NO: 1, comprising the HCDR1, the HCDR2 and theHCDR3 of

-   -   SEQ ID NOs: 16, 22 and 29, respectively;    -   SEQ ID NOs: 17, 23 and 30, respectively;    -   SEQ ID NOs: 16, 24 and 31, respectively;    -   SEQ ID NOs: 18, 25 and 32, respectively;    -   SEQ ID NOs: 19, 26 and 33, respectively;    -   SEQ ID NOs: 20, 27 and 34, respectively; or    -   SEQ ID NOs: 21, 28 and 35, respectively, and the LCDR1, the        LCDR2 and the LCDR3 of    -   SEQ ID NOs: 36, 43 and 51, respectively;    -   SEQ ID NOs: 37, 44 and 52, respectively;    -   SEQ ID NOs: 38, 45 and 53, respectively;    -   SEQ ID NOs: 39, 46 and 54, respectively;    -   SEQ ID NOs: 40, 47 and 55, respectively;    -   SEQ ID NOs: 41, 47 and 56, respectively;    -   SEQ ID NOs: 42, 48 and 57, respectively;    -   SEQ ID NOs: 37, 49 and 52, respectively; or    -   SEQ ID NOs: 37, 50 and 52, respectively.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 16, 22 and 29, respectively, andthe LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 36, 43 and 51,respectively; or the VH of SEQ ID NO: 58 and the VL of SEQ ID NO: 65.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 17, 23 and 30, respectively, andthe LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 37, 44 and 52,respectively; or the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 66.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 16, 24 and 31, respectively, andthe LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 38, 45 and 53,respectively; or the VH of SEQ ID NO: 60 and the VL or SEQ ID NO: 67.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 18, 25 and 32, respectively, andthe LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 39, 46 and 54,respectively; or the VH of SEQ ID NO: 61 and the VL or SEQ ID NO: 68.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 19, 26 and 33, respectively, andLCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 40, 47 and 55,respectively; or the VH of SEQ ID NO: 62 and the VL or SEQ ID NO: 69.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 20, 27 and 34, respectively, andthe LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 41, 47 and 56,respectively; or the VH of SEQ ID NO: 63 and the VL or SEQ ID NO: 70.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 21, 28 and 35, respectively, andthe LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 42, 48 and 57,respectively; or the VH of SEQ ID NO: 64 and the VL or SEQ ID NO: 71.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 17, 23 and 30, respectively, andLCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 37, 49 and 52,respectively; or the VH of SEQ ID NO: 59 and the VL or SEQ ID NO: 72.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 17, 23 and 30, respectively, andthe LCDR1, the LCDR2 and the LCDR3 of SEQ ID NOs: 37, 50 and 52,respectively; or the VH of SEQ ID NO: 59 and the VL or SEQ ID NO: 73.

In some embodiments, the antibody of the invention comprises the VH andthe VL wherein the VH comprises the amino acid sequence of SEQ ID NOs:58, 59, 60, 61, 62, 63 or 64.

In some embodiments, the antibody of the invention comprises the VH andthe VL, wherein the VL comprises the amino acid sequence of SEQ ID NOs:65, 66, 67, 68, 69, 70, 71, 72 or 73.

In some embodiments, the antibody of the invention comprises the VH ofSEQ ID NOs: 58, 59, 60, 61, 62, 63 or 64, and the VL of SEQ ID NOs: 65,66, 67, 68, 69, 70, 71, 72 or 73.

In some embodiments, the antibody of the invention comprises the HCDR1,HCDR2 and HCDR3 amino acid sequences of the VH of SEQ ID NOs: 65, 66,67, 68, 69, 70, 71, 72 or 73, and the LCDR1, LCDR2 and LCDR3 amino acidsequences of the VL of SEQ ID NOs: 65, 66, 67, 68, 69, 70, 71, 72 or 73,wherein the CDRs are defined according to Kabat, Chothia and/or IMGT.

Variants of the antibodies of the invention comprising the VH or the VLamino acid sequences shown in Table 8, Table 9 and Table 14 are withinthe scope of the invention. For example, variants may comprise one, two,three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen or fifteen amino acid substitutions in the VH and/orthe VL that do not adversely affect the antibody properties. In someembodiments, the sequence identity may be about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% to the VH or the VL amino acid sequence of theinvention.

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences (i.e., %identity=# of identical positions/total # of positions ×100), takinginto account the number of gaps, and the length of each gap, which needto be introduced for optimal alignment of the two sequences.

The percent identity between two amino acid sequences may be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences may be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www_gcg_com), using either a Blossum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

In some embodiments, the antibody of the invention comprises the VH thatis at least 90%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VH of SEQ ID NOs: 58, 59, 60, 61, 62, 63 or 64, whereinthe antibody exhibits one or more of the following properties:

-   -   an immune complex of the antibody and shCD154 does not activate        platelets, wherein platelet activation is measured by P-selectin        surface expression on platelets;    -   binds to CD154 with a dissociation constant (K_(D)) of about        5×10⁻⁹ M or less, when the K_(D) is measured using ProteOn™        XPR36 system using experimental design described in Example 1,        affinity measurements;    -   inhibits CD154-mediated human B cell proliferation with an IC₅₀        value of about 2.7×10⁻⁹ M or less; or    -   inhibits CD154-mediated expression of secreted embryonic        alkaline phosphatase (SEAP) under NF-κB-inducible interferon-β        (IFN-β) minimal promoter in HEK293 cells stably expressing SEAP        and human CD40 with an IC₅₀ value of about 2.1×10⁻⁸ M or less.

In some embodiments, the antibody of the invention comprises the VL thatis at least 90%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VL of SEQ ID NOs: 65, 66, 67, 68, 69, 70, 71, 72 or 73,wherein the antibody exhibits one or more of the following properties:

-   -   an immune complex of the antibody and shCD154 does not activate        platelets, wherein platelet activation is measured by P-selectin        surface expression on platelets; binds to CD154 with a        dissociation constant (K_(D)) of about 5×10⁻⁹ M or less, when        the K_(D) is measured using ProteOn™ XPR36 system using        experimental design described in Example 1, affinity        measurements;    -   inhibits CD154-mediated human B cell proliferation with an IC₅₀        value of about 2.7×10⁻⁹ M or less; or    -   inhibits CD154-mediated expression of secreted embryonic        alkaline phosphatase (SEAP) under NF-κB-inducible interferon-β        (IFN-β) minimal promoter in HEK293 cells stably expressing SEAP        and human CD40 with an IC₅₀ value of about 2.1×10⁻⁸ M or less.

In some embodiments, the antibody of the invention comprises the VH thatis at least 90%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VH of SEQ ID NOs: 58, 59, 60, 61, 62, 63 or 64 and theVL that is at least 90%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to the VL of SEQ ID NOs: 65, 66, 67, 68, 69, 70, 71, 72or 73, wherein the antibody exhibits one or more of the followingproperties:

-   -   an immune complex of the antibody and shCD154 does not activate        platelets, wherein platelet activation is measured by P-selectin        surface expression on platelets;    -   binds to CD154 with a dissociation constant (K_(D)) of about        5×10⁻⁹ M or less, when the K_(D) is measured using ProteOn™        XPR36 system using experimental design described in Example 1,        affinity measurements;    -   inhibits CD154-mediated human B cell proliferation with an IC₅₀        value of about 2.7×10⁻⁹ M or less; or    -   inhibits CD154-mediated expression of secreted embryonic        alkaline phosphatase (SEAP) under NF-κB-inducible interferon-β        (IFN-β) minimal promoter in HEK293 cells stably expressing SEAP        and human CD40 with an IC₅₀ value of about 2.1×10⁻⁸ M or less.

In some embodiments, the antibody of the invention comprises the VH thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VH of SEQ ID NOs: 58.

In some embodiments, the antibody of the invention comprises the VH thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VH of SEQ ID NOs: 59.

In some embodiments, the antibody of the invention comprises the VH thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VH of SEQ ID NOs: 60.

In some embodiments, the antibody of the invention comprises the VH thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VH of SEQ ID NOs: 61.

In some embodiments, the antibody of the invention comprises the VH thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VH of SEQ ID NOs: 62.

In some embodiments, the antibody of the invention comprises the VH thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VH of SEQ ID NOs: 63.

In some embodiments, the antibody of the invention comprises the VH thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VH of SEQ ID NOs: 64.

In some embodiments, the antibody of the invention comprises the VL thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VL of SEQ ID NOs: 65.

In some embodiments, the antibody of the invention comprises the VL thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VL of SEQ ID NOs: 66.

In some embodiments, the antibody of the invention comprises the VL thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VL of SEQ ID NOs: 67.

In some embodiments, the antibody of the invention comprises the VL thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VL of SEQ ID NOs: 68.

In some embodiments, the antibody of the invention comprises the VL thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VL of SEQ ID NOs: 69.

In some embodiments, the antibody of the invention comprises the VL thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VL of SEQ ID NOs: 70.

In some embodiments, the antibody of the invention comprises the VL thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VL of SEQ ID NOs: 71.

In some embodiments, the antibody of the invention comprises the VL thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VL of SEQ ID NOs: 72.

In some embodiments, the antibody of the invention comprises the VL thatis at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the VL of SEQ ID NOs: 73.

In some embodiments, the antibody the invention comprises the VH of SEQID NO: 58 and the VL or SEQ ID NO: 65, wherein the VH, the VL or boththe VH and the VL optionally comprise one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteenamino acid substitutions.

In some embodiments, the antibody of the invention comprises the VH ofSEQ ID NO: 59 and the VL or SEQ ID NO: 66, wherein the VH, the VL orboth the VH and the VL optionally comprise one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen orfifteen amino acid substitutions.

In some embodiments, the antibody of the invention comprises the VH ofSEQ ID NO: 60 and the VL or SEQ ID NO: 67, wherein the VH, the VL orboth the VH and the VL optionally comprise one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen orfifteen amino acid substitutions.

In some embodiments, the antibody of the invention comprises the VH ofSEQ ID NO: 61 and the VL or SEQ ID NO: 68, wherein the VH, the VL orboth the VH and the VL optionally comprise one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen orfifteen amino acid substitutions.

In some embodiments, the antibody of the invention comprises the VH ofSEQ ID NO: 62 and the VL or SEQ ID NO: 69, wherein the VH, the VL orboth the VH and the VL optionally comprise one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen orfifteen amino acid substitutions.

In some embodiments, the antibody of the invention comprises the VH ofSEQ ID NO: 63 and the VL or SEQ ID NO: 70, wherein the VH, the VL orboth the VH and the VL optionally comprise one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen orfifteen amino acid substitutions.

In some embodiments, the antibody of the invention comprises the VH ofSEQ ID NO: 64 and the VL or SEQ ID NO: 71, wherein the VH, the VL orboth the VH and the VL optionally comprise one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen orfifteen amino acid substitutions.

In some embodiments, the antibody of the invention comprises the VH ofSEQ ID NO: 59 and the VL or SEQ ID NO: 72, wherein the VH, the VL orboth the VH and the VL optionally comprise one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen orfifteen amino acid substitutions.

In some embodiments, the antibody of the invention comprises the VH ofSEQ ID NO: 59 and the VL or SEQ ID NO: 73, wherein the VH, the VL orboth the VH and the VL optionally comprise one, two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen orfifteen amino acid substitutions.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 amino acidsequences, shown in Table 8, Table 9 and Table 14, or conservativemodifications thereof, and wherein the antibodies retain the desiredfunctional properties of the antagonistic antibodies specificallybinding CD154.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 16, 22 and 29, respectively, andconservative modifications thereof.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 17, 23 and 30, respectively, andconservative modifications thereof.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 16, 24 and 31, respectively, andconservative modifications thereof.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 18, 25 and 32, respectively, andconservative modifications thereof

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 19, 26 and 33, respectively, andconservative modifications thereof.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of SEQ ID NOs: 20, 27 and 34, respectively, andconservative modifications thereof.

In some embodiments, the antibody of the invention comprises the HCDR1,the HCDR2 and the HCDR3 of EQ ID NOs: 21, 28 and 35, respectively, andconservative modifications thereof.

In some embodiments, the antibody of the invention comprises the LCDR1,the LCDR2, and the LCDR3 of SEQ ID NOs: 36, 43 and 51, respectively, andconservative modifications thereof.

In some embodiments, the antibody of the invention comprises the LCDR1,the LCDR2, and the LCDR3 SEQ ID NOs: 37, 44 and 52, respectively, andconservative modifications thereof.

In some embodiments, the antibody of the invention comprises the LCDR1,the LCDR2, and the LCDR3 SEQ ID NOs: 38, 45 and 53, respectively, andconservative modifications thereof.

In some embodiments, the antibody of the invention comprises the LCDR1,the LCDR2, and the LCDR3 SEQ ID NOs: 39, 46 and 54, respectively, andconservative modifications thereof.

In some embodiments, the antibody the invention comprises the LCDR1, theLCDR2, and the LCDR3 SEQ ID NOs: 40, 47 and 55, respectively, andconservative modifications thereof.

In some embodiments, the antibody of the invention comprises the LCDR1,the LCDR2, and the LCDR3 SEQ ID NOs: 41, 47 and 56, respectively, andconservative modifications thereof.

In some embodiments, the antibody of the invention comprises the LCDR1,the LCDR2, and the LCDR3 SEQ ID NOs: 42, 48 and 57, respectively, andconservative modifications thereof.

In some embodiments, the antibody of the invention comprises the LCDR1,the LCDR2, and the LCDR3 SEQ ID NOs: 37, 49 and 52, respectively, andconservative modifications thereof.

In some embodiments, the antibody of the invention comprises the LCDR1,the LCDR2, and the LCDR3 SEQ ID NOs: 37, 50 and 52, respectively, andconservative modifications thereof.

The antibodies of the invention comprising certain HCDR1, HCDR2, HCDR3,LCDR1, LCDR2 and LCDR3 sequences and conservative modifications thereofexhibit one or more of the following properties:

-   -   an immune complex of the antibody and shCD154 does not activate        platelets, wherein platelet activation is measured by P-selectin        surface expression on platelets; bind to CD154 with a        dissociation constant (K_(D)) of about 5×10⁻⁹ M or less, when        the K_(D) is measured using ProteOn™ XPR36 system using        experimental design described in Example 1, affinity        measurements;    -   inhibit CD154-mediated human B cell proliferation with an IC₅₀        value of about 2.7×10⁻⁹ M or less; or    -   inhibit CD154-mediated expression of secreted embryonic alkaline        phosphatase (SEAP) under NF-κB-inducible interferon-β (IFN-β)        minimal promoter in HEK293 cells stably expressing SEAP and        human CD40 with an IC₅₀ value of about 2.1×10⁻⁸ M or less.

“Conservative modification” refers to amino acid modifications that donot significantly affect or alter the binding characteristics of theantibody containing the amino acid sequences. Conservative modificationsinclude amino acid substitutions, additions and deletions. Conservativesubstitutions are those in which the amino acid is replaced with anamino acid residue having a similar side chain. The families of aminoacid residues having similar side chains are well defined and includeamino acids with acidic side chains (e.g., aspartic acid, glutamicacid), basic side chains (e.g., lysine, arginine, histidine), nonpolarside chains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine), uncharged polar side chains (e.g., glycine,asparagine, glutamine, cysteine, serine, threonine, tyrosine,tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan,histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine,valine, leucine, isoleucine, serine, threonine), amide (e.g.,asparagine, glutamine), beta-branched side chains (e.g., threonine,valine, isoleucine) and sulfur-containing side chains (cysteine,methionine). Furthermore, any native residue in the polypeptide may alsobe substituted with alanine, as has been previously described foralanine scanning mutagenesis (MacLennan et al., Acta Physiol. Scand.Suppl. 643:55-67, 1998; Sasaki et al., Adv. Biophys. 35:1-24, 1998).Amino acid substitutions to the antibodies of the invention may be madeby well-known methods for example by PCR mutagenesis (U.S. Pat. No.4,683,195). Alternatively, libraries of variants may be generated usingknown methods, for example using random (NNK) or non-random codons, forexample DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu,Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting antibody variants maybe tested for their characteristics using assays described herein.

Although the embodiments illustrated in the Examples comprise pairs ofvariable regions, one from a heavy chain and one from a light chain, askilled artisan will recognize that alternative embodiments may comprisesingle heavy or light chain variable regions. The single variable regionmay be used to screen for variable domains capable of forming atwo-domain specific antigen-binding fragment capable of for examplespecifically binding to CD154. The screening may be accomplished byphage display screening methods using for example hierarchical dualcombinatorial approach disclosed in Int. Pat. Publ. No. WO1992/01047 asdescribed herein.

Antibodies of the invention may be generated using various technologies.For example, the hybridoma method of Kohler and Milstein, Nature256:495, 1975 may be used to generate monoclonal antibodies. In thehybridoma method, a mouse or other host animal, such as a hamster, rator monkey, is immunized with human, marmoset or cyno CD154 or fragmentsof CD154, such as soluble form of CD154, followed by fusion of spleencells from immunized animals with myeloma cells using standard methodsto form hybridoma cells (Goding, Monoclonal Antibodies: Principles andPractice, pp. 59-103 (Academic Press, 1986)). Colonies arising fromsingle immortalized hybridoma cells are screened for production ofantibodies with desired properties, such as specificity of binding,cross-reactivity or lack thereof, and affinity for the antigen.

Various host animals may be used to produce the antibodies of theinvention. For example, Balb/c mice may be used to generate antibodies.The antibodies made in Balb/c mice and other non-human animals may behumanized using various technologies to generate more human-likesequences. Exemplary humanization techniques including selection ofhuman acceptor frameworks are known and include CDR grafting (U.S. Pat.No. 5,225,539), SDR grafting (U.S. Pat. No. 6,818,749), Resurfacing(Padlan, Mol Immunol 28:489-499, 1991), Specificity Determining ResiduesResurfacing (U.S. Pat. Publ. No. 20100261620), human-adaptation (orhuman framework adaptation) (U.S. Pat. Publ. No. US2009/0118127),Superhumanization (U.S. Pat. No. 7,709,226) and guided selection(Osbourn et al (2005) Methods 36:61-68, 2005; U.S. Pat. No. 5,565,332).In these methods, CDRs of parental antibodies are transferred onto humanframeworks that may be selected based on their overall homology to theparental frameworks, based on framework CDR length, homology orcanonical structure information, or a combination thereof.

Humanized antibodies may be further optimized to improve theirselectivity or affinity to a desired antigen by incorporating alteredframework support residues to preserve binding affinity (backmutations)by techniques such as those disclosed as described in Int. Pat. Publ.No. WO90/007861 and in Int. Pat. Publ. No. WO92/22653, or by introducingvariation to any of the CDRs to improve for example affinity of theantibody.

Transgenic mice carrying human immunoglobulin (Ig) loci in their genomemay be used to generate human antibodies against a target protein, andare described in for example Int. Pat. Publ. No. WO90/04036, U.S. Pat.No. 6,150,584, Int. Pat. Publ. No. WO99/45962, Int. Pat. Publ. No.WO02/066630, Int. Pat. Publ. No. WO02/43478, Lonberg et al (1994) Nature368:856-9; Green et al (1994) Nature Genet. 7:13-21; Green & Jakobovits(1998) Exp. Med. 188:483-95; Lonberg and Huszar (1995) Int. Rev.Immunol. 13:65-93; Bruggemann et al (1991) Eur. J. Immunol.21:1323-1326; Fishwild et al (1996) Nat. Biotechnol. 14:845-851; Mendezet al (1997) Nat. Genet. 15:146-156; Green (1999) J. Immunol. Methods231:11-23; Yang et al (1999) Cancer Res. 59:1236-1243; Bruggemann andTaussig (1997) Curr. Opin. Biotechnol. 8:455-458; Int. Pat. Publ. No.WO02/043478). The endogenous immunoglobulin loci in such mice may bedisrupted or deleted, and at least one complete or partial humanimmunoglobulin locus may be inserted into the mouse genome usinghomologous or non-homologous recombination, using transchromosomes, orusing minigenes. Companies such as Regeneron (www_regeneron_com),Harbour Antibodies (www_harbourantibodies_com), Open MonoclonalTechnology, Inc. (OMT) (www_omtinc_net), KyMab (www_kymab_com), Trianni(www.trianni_com) and Ablexis (www_ablexis_com) may be engaged toprovide human antibodies directed against a selected antigen usingtechnology as described above.

Human antibodies may be selected from a phage display library, where thephage is engineered to express human immunoglobulins or portions thereofsuch as Fabs, single chain antibodies (scFv), or unpaired or pairedantibody variable regions (Knappik et al., J. Mol Biol 296:57-86, 2000;Krebs et al., J Immunol Meth 254:67-84, 2001; Vaughan et al., NatureBiotechnology 14:309-314, 1996; Sheets et al., PITAS (USA) 95:6157-6162,1998; Hoogenboom and Winter, J Mol Biol 227:381, 1991; Marks et al., JMol Biol 222:581, 1991). The antibodies of the invention may be isolatedfor example from phage display library expressing antibody heavy andlight chain variable regions as fusion proteins with bacteriophage pIXcoat protein as described in Shi et al., J Mol Biol 397:385-96, 2010 andInt. Pat. Publ. No. WO09/085462). The libraries may be screened forphage binding to human, marmoset and/or cyno CD154 and the obtainedpositive clones may be further characterized, the Fabs isolated from theclone lysates, and expressed as full length IgGs. Such phage displaymethods for isolating human antibodies are described in for example:U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.;U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Pat. Nos.5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Pat. Nos.5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081 toGriffiths et al.

Preparation of immunogenic antigens and monoclonal antibody productionmay be performed using any suitable technique, such as recombinantprotein production. The immunogenic antigens may be administered to ananimal in the form of purified protein, or protein mixtures includingwhole cells or cell or tissue extracts, or the antigen may be formed denovo in the animal's body from nucleic acids encoding said antigen or aportion thereof.

The antibodies of the invention may be human or humanized.

In some embodiments, the antibody of the invention comprises a VHframework derived from human germline gene VH1_1-69, VH4_4-39, VH1_1-02or VH4_4-59.

In some embodiments, the antibody of the invention comprises a VLframework derived from human germline gene VKIV_B3, VKI_O12 or VL3_3R.

The antibodies of the invention may be of IgA, IgD, IgE, IgG or IgMtype. The antibodies of the invention may be of IgG1, IgG2, IgG3, IgG4type.

The antibodies of the invention may further be engineered to generatemodified antibody with similar or altered properties when compared tothe parental antibody. The VH, the VL, the VH and the VL, the constantregions, VH framework, VL framework, or any or all of the six CDRs maybe engineered in the antibodies of the invention.

The antibodies of the invention may be engineered by CDR grafting. Oneor more CDR sequences of the antibodies of the invention may be graftedto a different framework sequence. CDR grafting may be done usingmethods described herein. In some embodiments, the antibodies of theinvention comprise a VH that comprises the HDCR1 of SEQ ID NOs: 16, 17,18, 19, 20 or 21, the HCDR2 of SEQ ID NOs: 22, 23, 24, 25, 26, 27 or 28,the HCDR3 of SEQ ID NOs: 29, 30, 31, 32, 33, 34 or 35 and the VL thatcomprises the LCDR1 of SEQ ID NOs: 36, 37, 38, 39, 40, 41 or 42, theLCDR2 of SEQ ID NOs: 43, 44, 45, 6, 47, 48, 49 or 50 and/or the LCDR3 ofSEQ ID NOs: 51, 52, 53, 54, 55, 56 or 57, wherein the VH framework isnot derived from VH1_1-69, VH4_4-39, VH1_1-02 or VH4_4-59, and the VLframework is not derived from VKIV_B3, VKI_O12 or VL3_3R. The frameworksequences to be used may be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA and the encoded protein sequences for human heavyand light chain variable region genes can be found at IMGT, theinternational ImMunoGeneTics information System® www-imgt_org. Frameworksequences that may be used to replace the existing framework sequencesin the antibodies of the invention are those that show the highestpercent identity to C4LB5, C4LB89, C4LB94, C4LB150, C4LB189, C4LB191,C4LB199, C4LB231, C4LB232, C4LB35 and C4LB256.

The framework sequences of the parental and engineered antibodies mayfurther be modified, for example by backmutations to restore and/orimprove binding of the resulting antibody to the antigen as describedfor example in U.S. Pat. No. 6,180,370. The framework sequences of theparental and engineered antibodies may further be modified by mutatingone or more residues within the framework region, or even within one ormore CDR regions, to remove T-cell epitopes to thereby reduce thepotential immunogenicity of the antibody. This approach is also referredto as “deimmunization” and described in further detail in U.S. Pat.Publ. No. 20030153043.

The CDR residues of the antibodies of the invention may be mutated toimprove one or more binding properties of the antibody of interest.Site-directed mutagenesis or PCR-mediated mutagenesis may be performedto introduce the mutation(s) and the effect on antibody binding, orother functional property of interest, may be evaluated in in vitro orin vivo assays as described herein and provided in the Examples.Exemplary substitutions that may be introduced are conservativemodifications as discussed supra. Moreover, typically no more than one,two, three, four or five residues within a CDR region are altered.

Fc substitutions may be made to the antibody of the invention tomodulate antibody half-life. For example, one or more of thesubstitutions M252Y, S254T and T256E may be introduced to increase thehalf-life of the resulting antibody (Dall'Acqua et al., J Biol Chem281:23514-240, 2006).

Additionally, antibodies of the invention may be post-translationallymodified by processes such as glycosylation, isomerization,deglycosylation or non-naturally occurring covalent modification such asthe addition of polyethylene glycol moieties (pegylation) andlipidation. Such modifications may occur in vivo or in vitro. Forexample, the antibodies of the invention may be conjugated topolyethylene glycol (PEGylated) to improve their pharmacokineticprofiles. Conjugation may be carried out by techniques known to thoseskilled in the art. Conjugation of therapeutic antibodies with PEG hasbeen shown to enhance pharmacodynamics while not interfering withfunction (Knigh et al., Platelets 15:409-18, 2004; Leong et al.,Cytokine 16:106-19, 2001; Yang et al., Protein Eng. 16:761-70, 2003).

Antibodies or fragments thereof of the invention modified to improvestability, selectivity, cross-reactivity, affinity, immunogenicity orother desirable biological or biophysical property are within the scopeof the invention. Stability of an antibody is influenced by a number offactors, including (1) core packing of individual domains that affectstheir intrinsic stability, (2) protein/protein interface interactionsthat have impact upon the HC and LC pairing, (3) burial of polar andcharged residues, (4) H-bonding network for polar and charged residues;and (5) surface charge and polar residue distribution among other intra-and inter-molecular forces (Worn et al., J Mol Biol 305:989-1010, 2001).Potential structure destabilizing residues may be identified based uponthe crystal structure of the antibody or by molecular modeling incertain cases, and the effect of the residues on antibody stability maybe tested by generating and evaluating variants harboring mutations inthe identified residues. One of the ways to increase antibody stabilityis to raise the thermal transition midpoint (T_(m)) as measured bydifferential scanning calorimetry (DSC). In general, the protein T_(m)is correlated with its stability and inversely correlated with itssusceptibility to unfolding and denaturation in solution and thedegradation processes that depend on the tendency of the protein tounfold (Remmele et al., Biopharm 13:36-46, 2000). A number of studieshave found correlation between the ranking of the physical stability offormulations measured as thermal stability by DSC and physical stabilitymeasured by other methods (Gupta et al., AAPS PharmSci 5E8, 2003; Zhanget al., J Pharm Sci 93:3076-89, 2004; Maa et al., Int J Pharm140:155-68, 1996; Bedu-Addo et al., Pharm Res 21:1353-61, 2004; Remmeleet al., Pharm Res 15:200-8, 1997). Formulation studies suggest that aFab T_(m) has implication for long-term physical stability of acorresponding mAb.

In some embodiments, the antibody of the invention is a bispecificantibody.

In some embodiments, the antibody of the invention is a multispecificantibody.

The monospecific antibodies specifically binding CD154 of the inventionmay be engineered into bispecific antibodies which are also encompassedwithin the scope of the invention. The VL and/or the VH regions of theantibodies of the invention may be engineered using published methodsinto single chain bispecific antibodies as structures such as TandAb®designs (Int. Pat. Publ. No. WO1999/57150; U.S. Pat. Publ. No.2011/0206672) or into bispecific scFVs as structures such as thosedisclosed in U.S. Pat. No. 5,869,620; Int. Pat. Publ. No. WO1995/15388,1nt. Pat. Publ. No. WO1997/14719 or Int. Pat. Publ. No. W2011/036460.

The VL and/or the VH regions of the antibodies of the invention may beengineered into bispecific full length antibodies, where each antibodyarm binds a distinct antigen or epitope. Such bispecific antibodies maybe made by modulating the CH3 interactions between the two antibodyheavy chains to form bispecific antibodies using technologies such asthose described in U.S. Pat. No. 7,695,936; Int. Pat. Publ. No.WO2004/111233; U.S. Pat. Publ. No. 2010/0015133; U.S. Pat. Publ. No.2007/0287170; Int. Pat. Publ. No. WO2008/119353; U.S. Pat. Publ. No.2009/0182127; U.S. Pat. Publ. No. 2010/0286374; U.S. Pat. Publ. No.2011/0123532; Int. Pat. Publ. No. WO2011/131746; Int. Pat. Publ. No.WO2011/143545; or U.S. Pat. Publ. No. 2012/0149876.

For example, bispecific antibodies may be generated in vitro in acell-free environment by introducing asymmetrical mutations in the CH3regions of two monospecific homodimeric antibodies and forming thebispecific heterodimeric antibody from two parent monospecifichomodimeric antibodies in reducing conditions to allow disulfide bondisomerization according to methods described in Intl. Pat. Publ. No.WO2011/131746. In the methods, two monospecific bivalent antibodies areengineered to have certain substitutions at the CH3 domain that promoteheterodimer stability; the antibodies are incubated together underreducing conditions sufficient to allow the cysteines in the hingeregion to undergo disulfide bond isomerization; thereby generating thebispecific antibody by Fab arm exchange. The incubation conditions mayoptimally be restored to non-reducing. Exemplary reducing agents thatmay be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT),dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine(TCEP), L-cysteine and beta-mercaptoethanol, preferably a reducing agentselected from the group consisting of: 2-mercaptoethylamine,dithiothreitol and tris(2-carboxyethyl)phosphine. For example,incubation for at least 90 min at a temperature of at least 20° C. inthe presence of at least 25 mM 2-MEA or in the presence of at least 0.5mM dithiothreitol at a pH of from 5-8, for example at pH of 7.0 or at pHof 7.4 may be used.

Exemplary CH3 mutations that may be used in a first heavy chain and in asecond heavy chain of the bispecific antibody are K409R and/or F405L.

Additional bispecific structures into which the VL and/or the VH regionsof the antibodies of the invention may be incorporated are for exampleDual Variable Domain Immunoglobulins (DVD) (Int. Pat. Publ. No.WO2009/134776), or structures that include various dimerization domainsto connect the two antibody arms with different specificity, such asleucine zipper or collagen dimerization domains (Int. Pat. Publ. No.WO2012/022811, U.S. Pat. Nos. 5,932,448; 6,833,441). DVDs are fulllength antibodies comprising the heavy chain having a structureVH1-linker-VH2-CH and the light chain having the structureVL1-linker-VL2-CL; linker being optional.

The invention also provides for an antagonistic antibody thatspecifically binds CD154 of SEQ ID NO: 1 having certain VH and VLsequences, wherein the antibody VH is encoded by a first polynucleotideand the antibody VL is encoded by a second synthetic polynucleotide. Thepolynucleotide may be a complementary deoxynucleic acid (cDNA), and maybe codon optimized for expression in suitable host. Codon optimizationis a well-known technology.

In some embodiments, the polynucleotides encoding the antibody VH or VLof the invention comprise the sequences of SEQ ID NOs: 76, 77, 78 or 79.

(encoding VH of C4LB231) SEQ ID NO: 76GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCGGGCCAGCCAGAGCATCAGCAGCTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACTACGCCAACAGCCTGCAGAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCGACAGCATCCCCTGGACCTTCGGCCAG GGCACCAAGGTGGAAATCAAG(encoding VL of C4LB231) SEQ ID NO: 77CAGGTCCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCCGGCAGCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCGGCACCTTCAGCAGCTACGGCATCAGCTGGGTCCGACAGGCCCCAGGACAGGGCCTGGAATGGATGGGCTGGATCAGCCCCATCTTCGGCAACACCAACTACGCCCAGAAATTCCAGGGCAGAGTGACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGAAGCCGGTACTACGGCGACCTGGACTACTGGGGCCAGGGCACCCTGGTCACCGTGTC CTCT(encoding VH of C4LB191) SEQ ID NO: 78CAGGTGCAGCTGGTGCAGAGCGGCGCTCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCTGGCGCCAGCATGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCACCGACTACTACATCCACTGGGTGCGCCAGGCCCCAGGCCAGGGACTGGAATGGGTGGGACGGTTCAACCCCAACAGCGGCGACACCAACGGCGCCCAGAAATTCCAGGGCAGAGTGACCATGACCCGGGACACCAGCATCAGCACCGCCTACATGGAACTGACCCGGCTGCGGAGCGACGACACCGCCGTGTACCACTGTGCCAGAGAGGGCGAGCTGGCCGGCATCTTCTTCGACTACTGGGGCCAGGGCACCCTGGTGACAGTGTCCAGC (encoding VL of C4LB191)SEQ ID NO: 79 AGCTACGAGCTGACCCAGCCCCCCAGCGTGTCCGTGTCTCCTGGCCAGACCGCCAGCATCACCTGTAGCGGCGACAAGCTGGGCGACAAATACGTGTCCTGGAACCACCAGAAGCCCGGCCAGAGCCCCGTGCTGGTGATCTACCAGGACCGGAAGAGGCCCAGCGGCATCCCCGAGAGATTCAGCGGCAGCAACAGCGGCAACACCGCCACCCTGACCATCAGCGGCACCCAGGCCATGGACGAGGCCGACTACTACTGCCAGGCCTGGGACAGCAGCACCGTGGTGTTCGGCGGAGGC ACCAAGCTGACCGTGCTG

The invention also provides for an isolated polynucleotide encoding anyof the antibody heavy chain variable regions, the antibody light chainvariable regions, the antibody heavy chains and/or the antibody lightchains of the invention. Certain exemplary polynucleotides are disclosedherein, however, other polynucleotides which, given the degeneracy ofthe genetic code or codon preferences in a given expression system,encode the antibodies of the invention are also within the scope of theinvention. Exemplary polynucleotides are for example polynucleotideshaving the sequences shown in SEQ ID NOs: 76, 77, 78 AND 79. Thepolynucleotide sequences encoding the VH or the VL or a fragment thereofof the antibody of the invention may be operably linked to one or moreregulatory elements, such as a promoter or enhancer, that allowexpression of the nucleotide sequence in the intended host cell. Thepolynucleotide may be a cDNA.

The invention also provides for a vector comprising the polynucleotideof the invention. Such vectors may be plasmid vectors, viral vectors,vectors for baculovirus expression, transposon based vectors or anyother vector suitable for introduction of the synthetic polynucleotideof the invention into a given organism or genetic background by anymeans. For example, polynucleotides encoding light and/or heavy chainvariable regions of the antibodies of the invention, optionally linkedto constant regions, are inserted into expression vectors. The lightand/or heavy chains may be cloned in the same or different expressionvectors. The DNA segments encoding immunoglobulin chains may be operablylinked to control sequences in the expression vector(s) that ensure theexpression of immunoglobulin polypeptides. Such control sequencesinclude signal sequences, promoters (e.g. naturally associated orheterologous promoters), enhancer elements, and transcriptiontermination sequences, and are chosen to be compatible with the hostcell chosen to express the antibody. Once the vector has beenincorporated into the appropriate host, the host is maintained underconditions suitable for high level expression of the proteins encoded bythe incorporated polynucleotides.

Suitable expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors contain selection markerssuch as ampicillin-resistance, hygromycin-resistance, tetracyclineresistance, kanamycin resistance or neomycin resistance to permitdetection of those cells transformed with the desired DNA sequences.

Suitable promoter and enhancer elements are known in the art. Forexpression in a eukaryotic cell, exemplary promoters include lightand/or heavy chain immunoglobulin gene promoter and enhancer elements;cytomegalovirus immediate early promoter; herpes simplex virus thymidinekinase promoter; early and late SV40 promoters; promoter present in longterminal repeats from a retrovirus; mouse metallothionein-I promoter;and various art-known tissue specific promoters. Selection of theappropriate vector and promoter is well within the level of ordinaryskill in the art.

Large numbers of suitable vectors and promoters are known to those ofskill in the art; many are commercially available for generating asubject recombinant constructs. The following vectors are provided byway of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK,pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif.,USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia,Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG(Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

The invention also provides for a host cell comprising one or morevectors of the invention. “Host cell” refers to a cell into which avector has been introduced. It is understood that the term host cell isintended to refer not only to the particular subject cell but to theprogeny of such a cell, and also to a stable cell line generated fromthe particular subject cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not be identical to the parent cell, butare still included within the scope of the term “host cell” as usedherein. Such host cells may be eukaryotic cells, prokaryotic cells,plant cells or archeal cells.

Escherichia coli, bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species are examples of prokaryotic host cells. Othermicrobes, such as yeast, are also useful for expression. Saccharomyces(e.g., S. cerevisiae) and Pichia are examples of suitable yeast hostcells. Exemplary eukaryotic cells may be of mammalian, insect, avian orother animal origins. Mammalian eukaryotic cells include immortalizedcell lines such as hybridomas or myeloma cell lines such as SP2/0(American Type Culture Collection (ATCC®), Manassas, Va., CRL-1581), NSO(European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK,ECACC No. 85110503), FO (ATCC® CRL-1646) and Ag653 (ATCC® CRL-1580™)murine cell lines. An exemplary human myeloma cell line is U266 (ATTC®CRL-TIB-196™). Other useful cell lines include those derived fromChinese Hamster Ovary (CHO) cells such as CHO-K1SV (Lonza Biologics,Walkersville, Md.), CHO-K1 (ATCC® CRL-61™) or DG44.

The invention also provides for a method of producing an antibody of theinvention comprising culturing the host cell of the invention inconditions that the antibody is expressed, and recovering the antibodyproduced by the host cell. Methods of making antibodies and purifyingthem are well known in the art. Once synthesized (either chemically orrecombinantly), the whole antibodies, their dimers, individual lightand/or heavy chains, or other antibody fragments such as VH and/or VL,may be purified according to standard procedures, including ammoniumsulfate precipitation, affinity columns, column chromatography, highperformance liquid chromatography (HPLC) purification, gelelectrophoresis, and the like (see generally Scopes, ProteinPurification (Springer-Verlag, N.Y., (1982)). A subject antibody may besubstantially pure, e.g., at least about 80% to 85% pure, at least about85% to 90% pure, at least about 90% to 95% pure, or at least about 98%to 99%, or more, pure, e.g., free from contaminants such as cell debris,macromolecules, etc. other than the subject antibody.

The invention also provides for a method for producing an antagonisticantibody specifically binding CD154 of SEQ ID NO: 1, comprising:

-   -   incorporating the first polynucleotide encoding the VH of the        antibody and the second polynucleotide encoding the VL of the        antibody into an expression vector;    -   transforming a host cell with the expression vector;    -   culturing the host cell in culture medium under conditions        wherein the VL and the VH are expressed and form the antibody;        and    -   recovering the antibody from the host cell or culture medium.

The polynucleotides encoding certain VH or VL sequences of the inventionmay be incorporated into vectors using standard molecular biologymethods. Host cell transformation, culture, antibody expression andpurification are done using well known methods.

Methods of Treatment

Antagonistic antibodies specifically binding CD154 of the invention, forexample antibodies C4LB5, C4LB89, C4LB94, C4LB150, C4LB189, C4LB191,C4LB199, C4LB231, C4LB232, C4LB35 and C4LB236, may be used for thetreatment and/or prevention of any condition or disease whereinantagonizing the effects of CD154 may be therapeutically effective andmay reduce the symptoms of the disease. Examples thereof include thetreatment of allergic, autoimmune, cancer, transplant, GVHD,inflammatory and other conditions, especially conditions wherein theinduction of tolerance and/or the suppression of humoral immunity aretherapeutically desirable. Diseases that may be treated with theantibodies of the invention are immune-mediated inflammatory diseases orautoimmune diseases such as arthritis, systemic lupus erythematosus(SLE), inflammatory bowel disease, transplantation, kidneytransplantation, skin transplantation, bone marrow transplantation,graft versus host disease (GVHD), immune thrombocytopenia (ITP),multiple sclerosis, thyroiditis, type I diabetes or atherosclerosis.

Beyond simply blocking CD154-CD40 interactions, anti-CD154 therapy leadsto the induction of immunologic tolerance (Gordon et al., Diabetes 47:1199-1206, 1988); Markees et al., Transplantation 64: 329-335, 1997;Jarvinen et al., Transplantation 76: 1375-1379, 2003; Quezada et al.,Blood 102: 1920-1926, 2003; Frleta et al., J Immunother 26: 72-84, 2003;Elster et al., Transplantation 72: 1473-1478, 2001; Benda et al., CellTransplantation 11: 715-720, 2002; Wekerle and Sykes, Annual review ofmedicine 2001. 52: 353-370¹⁹; Camirand et al., Transplantation 73:453-461, 2002).

The invention also provides for a method of treating an immune-mediatedinflammatory disease or an autoimmune disease, comprising administeringa therapeutically effective amount of the antibody of the invention to asubject in need thereof for a time sufficient to treat theimmune-mediated inflammatory disease or autoimmune disease.

The invention also provides for a method of treating arthritis,comprising administering a therapeutically effective amount of theantibody of the invention to a subject in need thereof for a timesufficient to treat arthritis.

In some embodiments, arthritis is juvenile arthritis, rheumatoidarthritis, psoriatic arthritis, Reiter's syndrome, ankylosingspondylitis, or gouty arthritis.

The invention also provides for a method of treating lupus, comprisingadministering a therapeutically effective amount of the antibody of theinvention to a subject in need thereof for a time sufficient to treatlupus.

In some embodiments, lupus is systemic lupus erythematosus (SLE) orcutaneous lupus erythematosus (CLE).

In some embodiments, the subject has lupus nephritis.

The invention also provides for a method of treating inflammatory boweldisease, comprising administering a therapeutically effective amount ofthe antibody of the invention to a subject in need thereof for a timesufficient to treat inflammatory bowel disease.

In some embodiments, inflammatory bowel disease is Crohn's disease.

In some embodiments, inflammatory bowel disease is ulcerative colitis.

“Treatment” or “treat” refers to therapeutic treatment. Individuals inneed of treatment include those subjects diagnosed with the disorder ora symptom of the disorder. Subjects that may be treated also includethose prone to or susceptible to have the disorder, of those in whichthe disorder is to be prevented. Beneficial or desired clinical resultsinclude alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. Beneficial clinical result include, in a subject who hasreceived treatment, for example reduced proliferation of B cells ordendritic cells, reduction of inflammatory cytokines, adhesionmolecules, proteases, immunoglobulins (in instances where the CD40bearing cell is a B cell), combinations thereof, increased production ofanti-inflammatory proteins, a reduction in the number of autoreactivecells, an increase in immune tolerance, inhibition of autoreactive cellsurvival, and/or a decrease in one or more symptoms mediated bystimulation of CD40-expressing cells by CD154.

Clinical response may be assessed using screening techniques such asmagnetic resonance imaging (MRI) scan, x-radiographic imaging, computedtomographic (CT) scan, flow cytometry or fluorescence-activated cellsorter (FACS) analysis, histology, gross pathology, and blood chemistry,including but not limited to changes detectable by ELISA, RIA,chromatography, and the like.

Exemplary antibodies that may be used in the methods of the inventioncomprise the VH, the VL, the HCDR and/or the LCDR regions as shown inTable 2, Table 3, Table 4, Table 5, Table 6, table 7, Table 8, Table 9,Table 13 and Table 14, and antibodies C4LB5, C4LB89, C4LB94, C4LB150,C4LB189, C4LB191, C4LB199, C4LB231, C4LB232, C4LB35 and C4LB236.

The methods of the invention may be used to treat a subject belonging toany animal classification. Examples of subjects that may be treatedinclude mammals such as humans, rodents, dogs, cats and farm animals.

The antibodies of the invention may be useful in the preparation of amedicament for such treatment, wherein the medicament is prepared foradministration in dosages defined herein.

The antibodies of the invention may be administered in combination asecond therapeutic agent.

The second therapeutic agent may be any known therapy for autoimmune andinflammatory diseases, including any agent or combination of agents thatare known to be useful, or which have been used or are currently in use,for treatment of autoimmune and inflammatory diseases. Such therapiesand therapeutic agents include surgery or surgical procedures (e.g.splenectomy, lymphadenectomy, thyroidectomy, plasmapheresis,leukophoresis, cell, tissue, or organ transplantation, intestinalprocedures, organ perfusion, and the like), radiation therapy, therapysuch as steroid therapy and non-steroidal therapy, hormone therapy,cytokine therapy, therapy with dermatological agents (for example,topical agents used to treat skin conditions such as allergies, contactdermatitis, and psoriasis), immunosuppressive therapy, and otheranti-inflammatory monoclonal antibody therapy.

The second therapeutic agent may be a corticosteroid, an antimalarialdrug, an immunosuppressant, a cytotoxic drug, or a B-cell modulator.

In some embodiments, the second therapeutic agent is prednisone,prednisolone, methylprednisolone, deflazcort, hydroxychloroquine,azathioprine, methotrexate, cyclophosphamide, mycophenolate mofetil(MMF), mycophenolate sodium, cyclosporine, leflunomide, tacrolimus,RITUXAN® (rituximab), or BENLYSTA® (belimumab).

In some embodiments, the antibodies of the invention are administered incombination with a second therapeutic agent. Exemplary secondtherapeutic agents are corticosteroids, nonsteroidal anti-inflammatorydrugs (NSAIDs), salicylates, hydroxychloroquine, sulfasalazine,cytotoxic drugs, immunosuppressive drugs immunomodulatory antibodies,methotrexate, cyclophosphamide, mizoribine, chlorambucil, cyclosporine,tacrolimus (FK506; ProGrafrM), mycophenolate mofetil, and azathioprine(6-mercaptopurine), sirolimus (rapamycin), deoxyspergualin, leflunomideand its malononitriloamide analogs; anti-CTLA4 antibodies and Igfusions, anti-B lymphocyte stimulator antibodies (e.g., LYMPHOSTAT-BTM)and CTLA4-Ig fusions (BLyS-lg), anti-CD80 antibodies, anti-T cellantibodies such as anti-CD3 (OKT3), anti-CD4, corticosteroids such as,for example, clobetasol, halobetasol, hydrocortisone, triamcinolone,betamethasone, fluocinole, fluocinonide, prednisone, prednisolone,methylprednisolone; non-steroidal anti-inflammatory drugs (NSAIDs) suchas, for example, sulfasalazine, medications containing mesalamine (knownas 5-ASA agents), celecoxib, diclofenac, etodolac, fenprofen,flurbiprofen, ibuprofen, ketoprofen, meclofamate, meloxicam, nabumetone,naproxen, oxaprozin, piroxicam, rofecoxib, salicylates, sulindac, andtolmetin; phosphodiesterase-4 inhibitors, anti-TNFα antibodies REMICADE®(infliximab), SIMPONI® (golimumab) and HUMIRA® (adalimumab), thalidomideor its analogs such as lenalidomide.

The antibodies of the invention may be administered in combination witha second therapeutic agent simultaneously, sequentially or separately.

Treatment effectiveness or RA may be assessed using effectiveness asmeasured by clinical responses defined by the American College ofRheumatology criteria, the European League of Rheumatism criteria, orany other criteria. See for example, Felson et al. (1995) ArthritisRheum. 38: 727-35 and van Gestel et al. (1996) Arthritis Rheum. 39:34-40.

Administration/Pharmaceutical Compositions

The invention provides for pharmaceutical compositions of theantagonistic antibodies specifically binding CD154 of the invention anda pharmaceutically acceptable carrier. For therapeutic use, theantibodies the invention may be prepared as pharmaceutical compositionscontaining an effective amount of the antibody as an active ingredientin a pharmaceutically acceptable carrier. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the active compoundis administered. Such vehicles may be liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.For example, 0.4% saline and 0.3% glycine can be used. These solutionsare sterile and generally free of particulate matter. They may besterilized by conventional, well-known sterilization techniques (e.g.,filtration). The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, stabilizing, thickening,lubricating and coloring agents, etc. The concentration of the moleculesor antibodies of the invention in such pharmaceutical formulation mayvary widely, i.e., from less than about 0.5%, usually to at least about1% to as much as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% byweight and will be selected primarily based on required dose, fluidvolumes, viscosities, etc., according to the particular mode ofadministration selected. Suitable vehicles and formulations, inclusiveof other human proteins, e.g., human serum albumin, are described, forexample, in e.g. Remington: The Science and Practice of Pharmacy,21^(st) Edition, Troy, D. B. ed., Lipincott Williams and Wilkins,Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp691-1092, See especially pp. 958-989.

The mode of administration of the antibodies of the invention in themethods of the invention may be any suitable route such as parenteraladministration, e.g., intradermal, intramuscular, intraperitoneal,intravenous or subcutaneous, transmucosal (oral, intranasal,intravaginal, rectal) or other means appreciated by the skilled artisan,as well known in the art.

The antibodies of the invention may be administered to a subject by anysuitable route, for example parentally by intravenous (i.v.) infusion orbolus injection, intramuscularly or subcutaneously or intraperitoneally.i.v. infusion may be given over for, example, 15, 30, 60, 90, 120, 180,or 240 minutes, or from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

The dose given to a subject having an immune-mediated inflammatorydisease or an autoimmune disease such as rheumatoid arthritis issufficient to alleviate or at least partially arrest the disease beingtreated (“therapeutically effective amount”) and may be sometimes 0.005mg/kg to about 100 mg/kg, e.g. about 0.05 mg/kg to about 20 mg/kg orabout 0.1 mg/kg to about 20 mg/kg, or about 1 mg to about 20 mg/kg, orabout 4 mg/kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg, or,e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even higher,for example about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40,50, 60, 70, 80, 90 or 100 mg/kg.

A fixed unit dose may also be given, for example, 50, 100, 200, 500 or1000 mg, or the dose may be based on the patient's surface area, e.g.,500, 400, 300, 250, 200, or 100 mg/m². Usually between 1 and 8 doses,(e.g., 1, 2, 3, 4, 5, 6, 7 or 8) may be administered to treat theimmune-mediated inflammatory disease, such as rheumatoid arthritis, but9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may begiven.

The administration of the antibodies of the invention may be repeatedafter one day, two days, three days, four days, five days, six days, oneweek, two weeks, three weeks, one month, five weeks, six weeks, sevenweeks, two months, three months, four months, five months, six months orlonger. Repeated courses of treatment are also possible, as is chronicadministration. The repeated administration may be at the same dose orat a different dose. For example, the antibodies of the invention may beadministered at 0.1 mg/kg, at 1 mg/kg, at 5 mg/kg, at 8 mg/kg or at 16mg/kg at weekly interval for 8 weeks, followed by administration at 8mg/kg or at 16 mg/kg every two weeks for an additional 16 weeks,followed by administration at 8 mg/kg or at 16 mg/kg every four weeks byintravenous infusion.

The antibodies of the invention may be provided by maintenance therapy,such as, e.g., once a week for a period of 6 months or more.

For example, the antibodies of the invention may be provided as a dailydosage in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1,1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively,at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 after initiation of treatment, or any combinationthereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2hours, or any combination thereof.

The antibodies of the invention may also be administeredprophylactically in order to reduce the risk of developing theimmune-mediated inflammatory disease or an autoimmune disease such asarthritis or rheumatoid arthritis, and/or delay the onset of theimmune-mediated inflammatory disease of the autoimmune disease.

Thus, a pharmaceutical composition of the invention for intramuscularinjection may be prepared to contain 1 ml sterile buffered water, andbetween about 1 ng to about 100 mg/kg, e.g. about 50 ng to about 30mg/kg or more preferably, about 5 mg to about 25 mg/kg, of the antibodyof the invention.

For example, a pharmaceutical composition comprising the antibodies ofthe invention for intravenous infusion may be made up to contain about200 ml of sterile Ringer's solution, and about 8 mg to about 2400 mg,about 400 mg to about 1600 mg, or about 400 mg to about 800 mg of theantibodies of the invention for administration to a 80 kg patient.Methods for preparing parenterally administrable compositions are wellknown and are described in more detail in, for example, “Remington'sPharmaceutical Science”, 15th ed., Mack Publishing Company, Easton, Pa.

The “therapeutically effective amount” of the antibodies of theinvention effective in the treatment of an immune-mediated inflammatorydisease or an autoimmune disease may be determined by standard researchtechniques. For example, in vitro assays may be employed to helpidentify optimal dosage ranges. Optionally, the dosage of the antibodiesof the invention that may be effective in the treatment ofimmune-mediated inflammatory diseases or autoimmune diseases such asarthritis or rheumatoid arthritis may be determined by administering theantibodies to relevant animal models well known in the art. Selection ofa particular effective dose may be determined (e.g., via clinicaltrials) by those skilled in the art based upon the consideration ofseveral factors. Such factors include the disease to be treated orprevented, the symptoms involved, the patient's body mass, the patient'simmune status and other factors known by the skilled artisan. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the severity of disease, and should bedecided according to the judgment of the practitioner and each patient'scircumstances. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems. Theantibodies of the invention may be tested for their efficacy andeffective dosage using any of the models described herein.

The antibodies of the invention may be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional protein preparations andwell known lyophilization and reconstitution techniques can be employed.

Anti-Idiotypic Antibodies

The present invention provides for an anti-idiotypic antibody binding tothe antibody of the invention.

The invention also provides for an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 58 and the VL orSEQ ID NO: 65.

The invention also provides for an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 59 and the VL orSEQ ID NO: 66.

The invention also provides for an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 60 and the VL orSEQ ID NO: 67.

The invention also provides for an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 61 and the VL orSEQ ID NO: 68.

The invention also provides for an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 62 and the VL orSEQ ID NO: 69.

The invention also provides for an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 63 and the VL orSEQ ID NO: 70.

The invention also provides for an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 64 and the VL orSEQ ID NO: 71.

The invention also provides for an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 59 and the VL orSEQ ID NO: 72.

The invention also provides for an anti-idiotypic antibody specificallybinding the antibody comprising the VH of SEQ ID NO: 59 and the VL orSEQ ID NO: 73.

An anti-idiotypic (Id) antibody is an antibody which recognizes theantigenic determinants (e.g. the paratope or CDRs) of the antibody. TheId antibody may be antigen-blocking or non-blocking. Theantigen-blocking Id may be used to detect the free antibody in a sample(e.g. CD154 antibody of the invention described herein). Thenon-blocking Id may be used to detect the total antibody (free,partially bond to antigen, or fully bound to antigen) in a sample. An Idantibody may be prepared by immunizing an animal with the antibody towhich an anti-Id is being prepared.

An anti-Id antibody may also be used as an immunogen to induce an immuneresponse in yet another animal, producing a so-called anti-anti-Idantibody. An anti-anti-Id may be epitopically identical to the originalmAb, which induced the anti-Id. Thus, by using antibodies to theidiotypic determinants of a mAb, it is possible to identify other clonesexpressing antibodies of identical specificity. Anti-Id antibodies maybe varied (thereby producing anti-Id antibody variants) and/orderivatized by any suitable technique, such as those described elsewhereherein with respect to the antibodies specifically binding HLA-DRantibodies.

Immunoconjugates

An “immunoconjugate” refers to the antibody of the invention conjugatedto one or more heterologous molecule(s).

In some embodiments, the antibody of the invention is conjugated to oneor more cytotoxic agents. Exemplary such cytotoxic agents includechemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), and radioactiveisotopes.

In some embodiments, an immunoconjugate is an antibody-drug conjugate(ADC) in which the antibody of the invention is conjugated to one ormore drugs, such as to a maytansinoid (see, e.g., U.S. Pat. Nos.5,208,020, 5,416,06)); an auristatin such as monomethylauristatin drugmoieties DE and DF (MMAE and MMAF) (see, e.g., U.S. Pat. Nos. 5,635,483and 5,780,588, and 7,498,298), a dolastatin, a calicheamicin orderivative thereof (see, e.g., U.S. Pat. Nos. 5,712,374, 5,714,586,5,739, 116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296;Hinman et al., (1993) Cancer Res 53:3336-3342; and Lode et al., (1998)Cancer Res 58:2925-2928); an anthracycline such as daunomycin ordoxorubicin (see, e.g., Kratz et al., (2006) Current Med. Chem13:477-523; Jeffrey et al., (2006) Bioorganic & Med Chem Letters16:358-362; Torgov et al., (2005) Bioconj Chem 16:717-721; Nagy et al.,(2000) Proc Natl Acad Sci USA 97:829-834; Dubowchik et al, Bioorg. &Med. Chem. Letters 12: 1529-1532 (2002); King et al., (2002) J Med Chem45:4336-4343; and U.S. Pat. No. 6,630,579), methotrexate, vindesine, ataxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, andortataxel.

In some embodiments, the immunoconjugate comprises the antibody of theinvention conjugated to an enzymatically active toxin or fragmentthereof, such as diphtheria A chain, nonbinding active fragments ofdiphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricinA chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes.

In some embodiments, the antibody of the invention is conjugated to aradioactive atom to form a radioconjugate. A variety of radioactiveisotopes are available for the production of radioconjugates. Examplesinclude At211, 1131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212and radioactive isotopes of Lu. When the radioconjugate is used fordetection, it may comprise a radioactive atom for scintigraphic studies,for example tc99m or 1123, or a spin label for nuclear magneticresonance (NMR) imaging (also known as magnetic resonance imaging, mri),such as iodine-123 again, iodine-131, indium-I1, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of the antibody of the invention and the cytotoxic agent maybe made using a variety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HQ), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin maybe prepared as described in Vitetta et al., (1987) Science 238: 1098.Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See, e.g., WO94/11026.The linker may be a “cleavable linker” facilitating release of acytotoxic drug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., (1992) Cancer Res 52:127-131; U.S. Pat. No. 5,208,020) may be used.

The immunoconjugates or ADCs may be prepared with cross-linker reagentssuch as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB,SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS,sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A.).

The invention also provides for an immunoconjugate comprising theantibody specifically binding CD154 of SEQ ID NO: 1 of the inventionlinked to a therapeutic agent or an imaging agent.

The present invention will now be described with reference to thefollowing specific, non-limiting examples.

Example 1. Materials and Methods

Generation of Proteins Used

As endogenous CD154 signals as a trimer, recombinant CD154 was expressedin multiple ways to obtain a functional recombinant trimer. Solublehuman CD154 (shCD154; SEQ ID NO: 4), soluble Callithrix jacchus (commonmarmoset; herein referred to as marmoset) CD154 (smCD154; SEQ ID NO: 5)or soluble Macaca fascicularis (cynomolgous, herein referred to as cyno)CD154 (scCD154; SEQ ID NO: 6) were cloned and expressed as His6 (SEQ IDNO: 10) fusions (shCD154-his, SEQ ID NO: 7; smCD154-his, SEQ ID NO: 8;scCD154-his, SEQ ID NO: 9) or as a fusion with leucine zipper (ILZ) (SEQID NO: 11) (shCD154-ILZ, SEQ ID NO: 12; smCD154-ILZ, SEQ ID NO: 13;scCD154-ILZ, SEQ ID NO: 14). Cloning, expression and proteinpurification was done using standard methods. Both the His and ILZfusions were predominantly trimers. smCD154 and smCD154-ILZ werebiotinylated using EZ-Link™ Sulfo-NHS-LC-Biotin and Labeling Kit(Thermo, cat no 21327), the success of the biotinylation was analyzed byHABA-avidin assay (Thermo, Cat no 46610) and Octet. Cells expressinghuman CD40 (SEQ ID NO: 15) were used in some assays. D1.1 Jurkat cells(ATCC® CRL-10915™) endogenously expressing human CD154 were used in someassays.

Human CD154; SEQ ID NO: 1MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHG TGFTSFGLLKLMarmoset CD154; SEQ ID NO: 2MIETYNQPVPRSAATGPPVSMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEEKKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKPPNRFERILLRAANTHSSAKPCGQQSIHLGGIFELQPGASVFVNVTDPSQVSHG TGFTSFGLLKLCynomolgus CD154; SEQ ID NO: 3MIETYNQPSPRSAATGLPVRMKIFMYLLTIFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEEKKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHG TGFTSFGLLKL shCD154;SEQ ID NO: 4 MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL smCD154; SEQ ID NO: 5MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKPPNRFERILLRAANTHSSAKPCGQQSIHLGGIFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL scCD154; SEQ ID NO: 6MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL shCD154-his;SEQ ID NO: 7 GSHHHHHHGGGSMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHG TGFTSFGLLKLsmCD154-his; SEQ ID NO: 8GSHHHHHHGGGSMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKPPNRFERILLRAANTHSSAKPCGQQSIHLGGIFELQPGASVFVNVTDPSQVSHG TGFTSFGLLKLscCD154-his; SEQ ID NO: 9GSHHHHHHGGGSMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHG TGFTSFGLLKL His6;SEQ ID NO: 10 HHHHHH ILZ; SEQ ID NO: 11RMKQIEDKIEEILSKIYHIENEIARIKKLIGER shCD154-ILZ; SEQ ID NO: 12GSHHHHHHGGGSRMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGGSMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL smCD154-ILZ;SEQ ID NO: 13 GSHHHHHHGGGSRMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGGSMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKPPNRFERILLRAANTHSSAKPCGQQSIHLGGIFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL scCD154-ILZ;SEQ ID NO: 14 GSHHHHHHGGGSRMKQIEDKIEEILSKIYHIENEIARIKKLIGERGGGSMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL human CD40;SEQ ID NO: 15 MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQAffinity Measurements

Affinity measurements using Surface Plasmon Resonance (SPR) wereperformed using a ProteOn™ XPR36 system (BioRad). A biosensor surfacewas prepared by coupling anti-Human IgG Fc (Jackson cat #109-005-098) tothe modified alginate polymer layer surface of a GLC chip (BioRad, Cat#176-5011) using the manufacturer instructions for amine-couplingchemistry. Approximately 4700 RU (response units) of test antibodieswere immobilized. The kinetic experiments were performed at 25° C. inrunning buffer (DPBS+0.03% polysorbate P20+100 μg/ml BSA). To performkinetic experiments, 100 RU of antibodies were captured followed byinjections of analytes (shCD154-his and smCD154-his) at concentrationsranging from 0.391 nM to 100 nM (in a 4-fold serial dilution). Theassociation phase was monitored for 3 minutes at 50 μL/min followed by15 minutes of buffer flow (dissociation phase). The chip surface wasregenerated with two 18 second pulses of 100 mM H₃PO₄ (Sigma, Cat #7961)at 100 μL/min.

The collected data were processed using ProteOn™ Manager software.First, the data was corrected for background using inter-spots. Then,double reference subtraction of the data was performed by using thebuffer injection for analyte injections. The kinetic analysis of thedata was performed using a Langmuir 1:1 binding model.

CD154-Induced Ramos Cell Activation

Ability of the anti-CD154 antibodies to inhibit Ramos cell activationwas assessed using CD54 as a marker for cellular activation. Ramos cells(Burkitt's lymphoma cells, ATCC® CRL-1596™) maintained according tovendor protocol were seeded into a 96 well v-bottom plate at 2.0×10⁵cells/well in complete growth medium in 100 μl/well. The test antibodiesat concentrations 0.2, 2 or 20 μg/ml were pre-incubated with 40 ng/mlsmCD154-his for 1 h at room temperature (RT) and then added to thecells. The plate was covered and incubated overnight (37° C., 5% CO-2).On the following day the assay plate was spun down and the spenttreatment medium removed. The resulting cell pellets were washed withcold PBS/2% FBS and then cells stained with PE labelled anti-CD54(ICAM-1) antibody or appropriate isotype control for 1 h at 4° C. Thecells were washed with cold PBS/2% FBS, resuspended in 100 μl/well coldPBS/2% FBS and the fluorescent signal (yellow channel) measured on aflow cytometer. Antibodies were determined to be antagonists when theymet the criteria: % potency relative to 5C8 antibody >5% 5C8 potency,where % potency refers to the normalized percent inhibition relative to5C8 at the highest concentration tested.

NF-κB-SEAP Reporter Gene Assay

Ability of the anti-CD154 antibodies to inhibit CD154-induced CD40downstream signaling pathways were assessed using HEK-Blue™ CD40L cells(Invivogen), engineered to express human CD40 and transfected with asecreted embryonic alkaline phosphatase (SEAP) reporter gene under thecontrol of NF-κB-inducible promoter (IFN-β minimal promoter). The cellswere stimulated with either human or cyno CD154, or with Jurkat cells.HEK-Blue™ CD40L cells were maintained according to the vendor's protocoland all activity assays were performed in DMEM supplemented with 10%heat-inactivated fetal bovine serum and 1× Glutamax. The cells wereseeded into 96 well tissue culture plates at a cell density of 2.5 or5×10⁴ cells per well in 100 μl volume and incubated overnight (37° C.,5% CO₂). On the following day 4× solutions of shCD154-His orshCD154-ILZ, or D1.1 Jurkat cells were pre-incubated with 4× solutionsof anti-CD154 antibodies (at appropriate concentrations) at a 1:1 ratioto yield 2× solutions of CD154:antibody pre-complex mixtures. TheCD154:antibody mixtures were incubated at RT for 1 h, while the D1.1Jurkat:mAb mixture was incubated at 37° C. and 5% CO₂ for 1 h. At theend of the pre-complex incubation period, 100 μl/well of the 2×pre-complex solutions were added to the 96 well assay plate containingHEK-Blue™ CD40L cells; the final assay volume was 200 μl/well with finalCD154 concentrations of 80 ng/ml shCD154-His, or 40 ng/ml shCD154-ILZ,or 2.5-6.0×10⁴ D1.1 Jurkat cells. After 16-24 h of treatment time (37°C., 5% CO₂) the supernatants were analyzed for phosphatase (SEAP)activity by measuring absorbance (650 nm) of 40 μl/well of supernatantsthat was incubated with 160 μl/well of QUANTI-Blue™ (Invivogen) at 37°C. for 30-60.

Jurkat Cell-Mediated Dendritic Cell Activation Assay

Ability of the anti-CD154 antibodies to inhibit Jurkat-cell mediatedactivation of DC was evaluated by measuring reduced production ofvarious cytokines by the DC. Human monocytes (Biologic Specialties) werecultured with 50 ng/ml IL-4 and GM-CSF for six days. The cells werereplenished with fresh media (with IL-4 and GM-CSF) on day 3. Theimmature DCs (iDCs) (CD1a⁺CD14^(low) CD83⁻) were used in cell assays onday 6. 2.5×10⁵ D1.1 Jurkat cells (irradiated at 1000 rads) wereincubated with 0.000064-25 μg/ml μg/ml anti-CD154 antibodies for 15-20minutes then co-cultured with 2.5×10⁴ iDCs in a final volume of 200μl/well in a 96-well round bottom plate. After 48 hour incubationsupernatants were harvested for cytokine analysis.

Jurkat Cell-Mediated B Cell Activation Assay

Ability of the anti-CD154 antibodies to inhibit Jurkat-cell mediated Bcell activation was evaluated by assessing the effect of the antibodieson B cell proliferation. 1×10⁵ D1.1 Jurkat cells (irradiated at 5000rads) were co-cultured with 1×10⁵ human tonsil B cells in the presenceof IL-21 (100 ng/ml) and 0.0077 ng/ml-15 μg/ml of anti-CD154 antibodiesin a final volume of 200 μl/well in a 96-well round bottom plate. After2 days incubation methyl (-3H)-Thymidine (0.5 μCi/well) were added tothe cultures and human B cell proliferation was determined afterovernight incubation.

CD154-Mediated B Cell Activation Assay

Ability of the anti-CD154 antibodies to inhibit recombinantCD154-mediated B cell activation was evaluated inhuman or cynomolgus Bcells. 1×10⁵ human tonsil B cells or cynomolgus monkey spleen cells werecultured with 100 ng/ml rhIL-21, 0.5 μg/ml shCD154-ILZ, and 0.0077ng/ml-15 μg/ml of anti-CD154 antibodies in a final volume of 200 μl/wellin a 96-well round bottom plate. After 2 days incubation methyl(-3H)-Thymidine (0.5 μCi/well) were added to the cultures and human Bcell proliferation was determined after overnight incubation.

Example 2. Isolation of Anti-CD154 Antibodies from Phage DisplayLibraries

CD154 binding Fabs were selected from de novo pIX phage displaylibraries as described in Shi et al., J Mol Biol 397:385-96, 2010; Int.Pat. Publ. No. WO2009/085462; U.S. Pat. Publ. No. US2010/0021477).Briefly, the libraries were generated by diversifying human scaffoldswhere germline VH genes IGHV1-69*01, IGHV3-23*01, and IGHV5-51*01 wererecombined with the human IGHJ-4 minigene via the H3 loop, and humangermline VLkappa genes O12 (IGKV1-39*01), L6 (IGKV3-11*01), A27(IGKV3-20*01), and B3 (IGKV4-1*01) were recombined with the IGKJ-1minigene to assemble complete VH and VL domains. The positions in theheavy and light chain variable regions around H1, H2, L1, L2 and L3loops corresponding to positions identified to be frequently in contactwith protein and peptide antigens were chosen for diversification.Sequence diversity at selected positions was limited to residuesoccurring at each position in the IGHV or IGLV germline gene families ofthe respective IGHV or IGLV genes. Diversity at the H3 loop wasgenerated by utilizing short to mid-sized synthetic loops of lengths7-14 amino acids. The amino acid distribution at H3 was designed tomimic the observed variation of amino acids in human antibodies. Librarydesign is detailed in Shi et al., J Mol Biol 397:385-96, 2010. Thescaffolds utilized to generate libraries were named according to theirhuman VH and VL germline gene origin. The three heavy chain librarieswere combined with the four germline light chains or germline lightchain libraries to generate 12 unique VH:VL combinations for panningexperiments against smCD154 or cells expressing full-length cyno CD154.

The libraries were panned against either full length cyno CD154 (SEQ IDNO: 3) expressed stably in CHO-s cells or biotinylated andnonbiotinylated smCD154 (SEQ ID NO: 5). After several rounds of panning,a polyclonal phage ELISA using smCD154 as antigens was performed todetect the specific enrichment of individual panning experiments. Thephage collected from those panning experiments which demonstratedenrichment for binders to smCD154 were further screened with amonoclonal Fab ELISA in which Fab proteins expressed from individual Fabclones were used as binders to nonbiotinylated smCD154 directly coatedon the plate. The Fab clones with binding signal four times higher thanthe negative control Fabs were selected to be screened in full IgGformat. Select Fabs were cloned into IgG2sigma/κappa backbone andcharacterized further using for binding to D1.1 Jurkat cells. IgG2sigmais a silent Fc and has substitutions V234A, G237A, P238S, H268A, V309L,A330S and P331S when compared to the wild type IgG2. IgG2sigma isdescribed in U.S. Pat. No. 8,961,967.

Example 3. Generation of Anti-CD154 Antibodies in Rats

Anti-CD154 antibodies were generated using transgenic rats expressinghuman immunoglobulin loci, the OmniRat®; OMT, Inc. The OmniRat®endogenous immunoglobulin loci are replaced by human Igκ and Igλ lociand a chimeric human/rat IgH locus with V, D and J segments of humanorigin linked to the rat C_(H) locus. The IgH locus contains 22 humanV_(H)s, all human D and J_(H) segments in natural configuration linkedto the rat C_(H) locus. Generation and characterization of the OmniRat®is described in Osborn, et al. J Immunol 190: 1481-1490, 2013; and Int.Pat. Publ. No. WO2014/093908.

OmniRat® was immunized with smCD154 by the repetitive immunization atmultiple sites (RIMMS) protocol. Following a 45 day immunizationregimen, lymph nodes were harvested from all four rats and used togenerate hybridomas. Hybridoma supernatants in 96-well plates werescreened via binding ELISA to identify mAbs which exhibited binding tosmCD154, from which hybridoma supernatants exhibiting an assay signalgreater than 3-fold the negative control average were selected.

Select antibodies were cloned as full length IgG2sigma/k. Antibodiesdemonstrating antagonist activity in CD154-induced Ramos cell activationwere selected for further characterization.

Example 4. Characterization of the Antibodies

Several anti-CD154 antibodies obtained from phage display or transgenicanimals expressing human immunoglobulin loci which demonstratedantagonistic activity as described in Examples 2 and 3 were sequencedand further characterized for their binding to human and cyno dendriticcells, for their ability to inhibit human and cyno dendritic and B cellfunctions, and for antibody effector functions. The VH and the VLregions of the antibodies were sequenced using standard methods.

Table 2 shows the HCDR1 amino acid sequences of select antibodies.

Table 3 shows the HCDR2 amino acid sequences of select antibodies.

Table 4 shows the HCDR3 amino acid sequences of select antibodies.

Table 5 shows the LCDR1 amino acid sequences of select antibodies.

Table 6 shows the LCDR2 amino acid sequences of select antibodies.

Table 7 shows the LCDR3 amino acid sequences of select antibodies.

Table 8 shows the VH amino acid sequences of select antibodies.

Table 9 shows the VL amino acid sequences of select antibodies

TABLE 2 HCDR1 SEQ ID mAb ID Sequence NO: C4LB5 SYAIS 16 C4LB89 SYGIS 17C4LB94 SYAIS 16 C4LB150 SYSFYWG 18 C4LB189 AYYIH 19 C4LB191 DYYIH 20C4LB199 SFIYYWG 21

TABLE 3 HCDR2 SEQ ID mAb ID Sequence NO: C4LB5 GIIPIFGTANYAQKFQG 22C4LB89 WISPIFGNTNYAQKFQG 23 C4LB94 GISPYFGNTNYAQKFQG 24 C4LB150SLYYSGSTYYNPSLKS 25 C4LB189 RINPDSGGTDYAQRFQG 26 C4LB191RFNPNSGDTNGAQKFQG 27 C4LB199 CIYSSGGTYYNPSLKS 28

TABLE 4 HCDR3 SEQ ID mAb ID Sequence NO: C4LB5 GASVWDGPAEVFDY 29 C4LB89SRYYGDLDY 30 C4LB94 DTGWVGAFYLDY 31 C4LB150 LQLGTTTDYFDH 32 C4LB189DWNYYDGSGYFGPGYYGLDV 33 C4LB191 EGELAGIFFDY 34 C4LB199 LWLGTTTDYFDY 35

TABLE 5 LCDR1 SEQ ID mAb ID Sequence NO: C4LB5 KSSQSVLASSNNENFLA 36C4LB89 RASQSISSYLN 37 C4LB94 KSSQSVLYSSNNKNYLA 38 C4LB150 SGDELGDKFAC 39C4LB189 SGDKLGDKYVC 40 C4LB191 SGDKLGDKYVS 41 C4LB199 SGDKLGDKFAC 42

TABLE 6 LCDR2 SEQ ID mAb ID Sequence NO: C4LB5 SASTRES 43 C4LB89 YANSLQS44 C4LB94 WASTRES 45 C4LB150 QENKRPS 46 C4LB189 QDRKRPS 47 C4LB191QDRKRPS 47 C4LB199 QDDKRPS 48

TABLE 7 LCDR3 SEQ ID mAb ID Sequence NO: C4LB5 QQAYTTPFT 51 C4LB89QQSDSIPWT 52 C4LB94 QQYYSTPLT 53 C4LB150 QAWDSDTAV 54 C4LB189 QAWDSGTVV55 C4LB191 QAWDSSTVV 56 C4LB199 QAWDSNTVV 57

TABLE 8 VH SEQ ID mAb ID VH name Sequence NO: C4LB5 C4LH12QVQLVQSGAEVKKPGSSVKVSCKASGGTFS 58 SYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTA VYYCARGASVWDGPAEVFDYWGQGTLVTV SS C4LB89C4LH165 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS 59 SYGISWVRQAPGQGLEWMGWISPIFGNTNYAQKFQGRVTITADESTSTAYMELSSLRSEDT AVYYCARSRYYGDLDYWGQGTLVTVSS C4LB94C4LH99 QVQLVQSGAEVKKPGSSVKVSCKASGGTFS 60 SYAISWVRQAPGQGLEWMGGISPYFGNTNYAQKFQGRVTITADESTSTAYMELSSLRSEDT AVYYCARDTGWVGAFYLDYWGQGTLVTVS S C4LB150C4LH201 QLQLQESGPGLVKPSETLSLTCTVSGGSISS 61YSFYWGWIRQPPGQGLEWIGSLYYSGSTYY NPSLKSRATMSVVTSKTQFSLNLNSVTAADTAVYYCARLQLGTTTDYFDHWGQGTLVTVSS C4LB189 C4LH240QVQLVQSGAEVKKPGASVKVSCKASGYTFA 62 AYYIHWVRQAPGQGLEWMGRINPDSGGTDYAQRFQGRVTMTRDTSISTAYMELSRLRSD DTAVFYCARDWNYYDGSGYFGPGYYGLDV WGQGTTVTVSSC4LB191 C4LH242 QVQLVQSGAEVKKPGASMKVSCKASGYTFT 63DYYIHWVRQAPGQGLEWVGRFNPNSGDTN GAQKFQGRVTMTRDTSISTAYMELTRLRSDDTAVYHCAREGELAGIFFDYWGQGTLVTVS S C4LB199 C4LH250QVQLQESGPGLVKPSETLSLTCTVSGDSISS 64 FIYYWGWIRQPPGKGLDWVGCIYSSGGTYYNPSLKSRVTISVDTSKNQFSLKLPSVTAADT AVYYCARLWLGTTTDYFDYWGQGTLVTVSS

TABLE 9 VL SEQ ID mAb ID VL Sequence NO: C4LB5 C4LL8DIVMTQSPDSLAVSLGERATINCKSSQSVLA 65 SSNNENFLAWYQQKPGQPPKWYSASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQAYTTPFTFGQGTKVEIK C4LB89 C4LL49DIQMTQSPSSLSASVGDRVTITCRASQSISS 66 YLNWYQQKPGKAPKLLIYYANSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSD SIPWTFGQGTKVEIK C4LB94 PH9L2DIVMTQSPDSLAVSLGERATINCKSSQSVLY 67 SSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY YCQQYYSTPLTFGQGTKVEIK C4LB150 C4LL82SYELTQPPSVSVSPGQTASITCSGDELGDKF 68 ACWYQQKPGQSPVLVIWQENKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWD SDTAVFGGGTKLTVL C4LB189 C4LL116SYELTQPPSVSVSPGQTASVTCSGDKLGDK 69 YVCWYQRKPGQSPVLVIYQDRKRPSGIPERFSGSNSGNTATLTISGTQAIDEADYYCQAWD SGTVVFGRGTKLTVL C4LB191 IAPL39SYELTQPPSVSVSPGQTASITCSGDKLGDKY 70 VSWNHQKPGQSPVLVIYQDRKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWD SSTVVFGGGTKLTVL C4LB199 C4LL125SYELTQPPSVSVSPGQTVSITCSGDKLGDKF 71 ACWYQQKPGQSPVVVIYQDDKRPSGIPERFSGSTSGNTATLTISGTQAMDEADYYCQAWD SNTVVFGGGTKLTVL

The antibodies inhibited function of both endogenous CD154 provided onJurkat cells (D1.1 Jurkat cells in Table 10) and recombinantly expressedhuman CD154 trimer (expressed as shCD154-ILZ or shCD154-his) as measuredusing the NF-κB SEAP reporter gene assay. The antibodies inhibitedsignaling with IC₅₀ values ranging from 0.08-21.15 nM. The IC₅₀ valuesfor select antibodies in the assay are shown in Table 10. The range ofIC₅₀ values in the table for each antibody represent the lowest and thehighest IC₅₀ values obtained from separate experiments, while thesingular value indicates the antibody was tested in one experiment, oronly one valid IC₅₀ value was available.

TABLE 10 NF-κB SEAP reporter gene assay; IC₅₀ (nM) mAb ID D1.1. Jurkat*shCD154-ILZ* shCD154-his* C4LB5 1.55 0.54 1.03-1.37 C4LB89 0.08-0.321.91-2.44 3.93-5.69 C4LB94 0.13 4.37  7.57-21.15 C4LB150 3.57 4.958.87-9.81 C4LB189 2.06 1.63  6.51-13.60 C4LB191 2.19-3.46 C4LB199 1.211.09 2.00-2.70 *CD154 format used to induce signaling

The ability of the antibodies to inhibit dendritic cell activation wasassessed using IL-12p40 secretion as a marker for DC activation. Theantibodies inhibited DC activation induced by endogenous CD154 providedon Jurkat cells with IC₅₀ values ranging from 0.02 to 0.49 nM. Table 11shows the IC₅₀ values for select antibodies in the assay. The IC₅₀ rangein the Table represents the lowest and the highest IC₅₀ value obtainedin separate experiments across 2-4 donors with 1-6 repetitions.

TABLE 11 Jurkat cell-mediated dendritic cell activation assay; mAb IDIC₅₀ (nM) C4LB5 0.32-0.49 C4LB89 0.02-0.09 C4LB94 0.07-0.09 C4LB1500.25-0.30 C4LB189 0.15-0.16 C4LB191 0.38-0.39 C4LB199 0.19-0.20

The ability of the antibodies to inhibit human or cyno B cell activationwas measured using B cell proliferation as readout. The antibodiesinhibited both endogenous CD154 (D1.1 Jurkat cells) and recombinanthuman CD154 trimer (shCD-54-IZ) induced proliferation with IC₅₀ valuesranging from 0.01-5.35 nM. Table 12 shows the C₅₀ values for variousantibodies obtained in human or cyno B cell activation assay. The IC₅₀range in the table represents the lowest and the highest IC₅₀ valueobtained in separate experiments across 2-4 donors with 1-6 repetitions.A singular IC₅₀ value in the Table indicates availability of one validIC₅₀ value.

TABLE 12 B cell activation assay; IC₅₀ (nM) D1.1 Jurkat cells/shCD154-ILZ/ shCD154-ILZ/ human human cyno mAb ID B cells* B cells* Bcell* C4LB5 0.19-0.28 2.74 5.35 C4LB89 0.01-0.02 0.20-0.48 0.13-0.44C4LB94 0.01-0.03 0.07-0.14 0.30-0.31 C4LB150 0.27-0.31 nd 1.14-2.00C4LB189 0.47-0.65 nd 4.35-5.16 C4LB191 0.64-1.00 nd 0.25-0.36 C4LB1990.04-0.27 nd 1.89-2.31 nd: not done *CD154 format used to inducesignaling/source of B cells

Example 5. Engineering of Antibodies to Minimize Post-TranslationalModification Risk

The VL of antibody C4LB89 contained a putative deamination site in LCDR2(N52-S53 in the light chain C4LL49, SEQ ID NO: 66). Substitutions wereindividually made to each position (N52S and S53T) in the VL. Themutated light chains were co-expressed with the parental heavy chainC4LH165 (SEQ ID NO: 59) to generate antibodies C4LB235 and C4LB236 asIgG2sigma/κ. The amino acid sequences of the LCDR2 and VL of C4LB235 andC4LB236 are shown in Table 13 and Table 14, respectively. C4LB235comprises the HCDRs of SEQ ID NOs: 17, 23 and 30, the LCDRs of SEQ IDNOs 37, 49 and 52, the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 72.C4LB236 comprises the HCDRs of SEQ ID NOs: 17, 23 and 30, the LCDRs ofSEQ ID NOs 37, 50 and 52, the VH of SEQ ID NO: 59 and the VL of SEQ IDNO: 73.

TABLE 13 LCDR2 SEQ ID mAb ID Sequence NO: C4LB235 YASSLQS 49 C4LB236YANTLQS 50

TABLE 14 VL SEQ ID mAb ID VL Sequence NO: C4LB235 C4LL160DIQMTQSPSSLSASVGDRVTITCR 72 ASQSISSYLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQSDSIP WTFGQGTKVEIK C4LB236C4LL161 DIQMTQSPSSLSASVGDRVTITCR 73 ASQSISSYLNWYQQKPGKAPKLLIYYANTLQSGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQSDSIP WTFGQGTKVEIK

Both antibodies were tested for their ability to inhibit cyno B cellproliferation. C4LB235 showed similar potency to C4LB89, while C4LB236showed reduced potency compared to C4LB89.

Example 6. Effector Silent Anti-CD154 Antibodies do not Induce PlateletActivation

Anti-CD154 antibodies have been developed in the clinic with positiveoutcomes in patients with autoimmune diseases, however, due to incidentsof thromboembolism (TE) further clinical development of the antibodieswere halted. Humanized 5c8 antibody (IgG1/κ) is anti-CD154 antibodywhich in the clinic induced TE (Yazdany et al., Lupus 13:377-380, 2004).It is hypothesized that thromboembolism (TE) mediated by humanized 5c8is a result of platelet activation and aggregation from formation ofhigh-ordered anti-CD154/CD154 immune complexes (IC) that cross-linkplatelets by binding Fc to platelet FcγRIIa receptor. In vitro,engineered 5c8 antibody with silenced Fc (D265A substitution in IgG1)lacking FcγRIIa receptor binding did not activate platelets (Xie et al.,J Immunol 192:4083-4092, 2014).

Anti-CD154 antibodies with abolished binding to at least FcγRIIa andhaving reduced effector functions may thus be more suitable as atherapeutic with a reduced risk for TE.

To that end, effector silent anti-CD154 antibodies were generated withvarious Fc substitutions and tested for their effect on plateletactivation.

VH and VL of the humanized 5c8 antibody (Karpusas et al., Structure 9:321-329, 2001) were cloned as IgG1sigma/κ, IgG1sigmaYTE/κ, IgG2sigma/κor IgG2sigmaYTE/κ to evaluate effect of the Fe on platelet activation;the resulting antibodies were named 5c8IgG1sigma, 5c8IgG1sigmaYTE,5c8IgG2sigma and 5c8IgG2sigmaYTE). IgG1sigma contains substitutionsL234A, L235A, G237A, P238S, H268A, A330S, and P331S when compared to thewild type IgG1. IgG1sigmaYTE contains L234A, L235A, G237A, P238S, M252Y,S254T, T256E H268A, A330S and P331S substitutions. IgG2sigma containsV234A, G237A, P238S, H268A, V309L, A330S and P331S substitutions.IgG2sigmaYTE contains V234A, G237A, P238S, M252Y, S254T, T256E, H268A,V309L, A330S and P331S substitutions. Residue numbering is according tothe EU Index. Antibodies with IgG2sigma backbone lack effector functionsand binding to FcγR as described in U.S. Pat. No. 8,961,967. YTEsubstitution (M252Y, S254T, T256E) is described in Dall'Acqua et al., JBiol Chem 281:23514-24, 2006.

VH and the VL domains of the humanized 5c8 are shown in SEQ ID NOs: 74and 75, respectively.

SEQ ID NO: 74 QVQLVQSGAEVVKPGASVKLSCKASGYIFTSYYMYWVKQAPGQGLEWIGEINPSNGDTNFNEKFKSKATLTVDKSASTAYMELSSLRSEDTAVYYC TRSDGRNDMDSWGQGTLVTVSSSEQ ID NO: 75 DIVLTQSPATLSVSPGERATISCRASQRVSSSTYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLTISSVEPEDFATYYCQHSW EIPPTFGGGTKLEIK

5c8IgG1sigma, 5c8IgG1sigmaYTE, 5c8IgG2sigma and 5c8IgG2sigmaYTE weretested for their effect on platelet activation.

Blood from healthy human donors, pre-screened for low response toshCD154 alone, was used. Platelet activation was evaluated by flowcytometry using validated platelet activation markers PAC-1 (activatedGPIIb/IIIa) and CD62p (P-selectin). Briefly, whole blood (WB) was addedto modified Tyrodes-HEPES buffer containing 1 mM CaCl₂, and anti-PAC1and anti-CD62p antibodies with or without FcγRIIa blocking antibody(clone IV.3, StemCell Technologies #60012) were added to the mixture andincubated for 25 minutes. Pre-formed immune complexes of soluble CD154(PeproTech, cat #310-02; SEQ ID NO: 4 or Tonbo Biosciences, cat#21-7088)/antibody at molar ratio of 3:1 CD154:anti-CD154 were added tothe mixture and incubated for another 20 minutes; platelets were fixedin 1% formalin followed by FACS analysis. Platelet activation for eachcondition was assessed as % of gated platelets (CD61 positive events)expressing PAC-1 and CD62p; 5000 CD61 expressing events (platelets) werecaptured and analyzed for each treatment condition. Results of theexperiment are shown in FIG. 1. CD154/5c8IgG1 (wild-type IgG1) ICactivated platelets, while CD154 IC with 5c8-IgG1sigma, 5c8IgG1sigmaYTE,5c8IgG2sigma and 5c8IgG2sigma-YTE did not activate platelets. Plateletactivation with ADP was not inhibited by the immune complexes (data notshown). None of the antibodies alone activated platelets (data notshown).

Anti-CD154 antibodies C4LB5, C4LB89, C4LB94, C4LB150, C4LB189, C4LB191,C4LB199 (all IgG2sigma effector silent mAbs) were also tested inplatelet activation assay to confirm the antibodies had abolishedbinding to FcγRIIa and did not activate platelets. FIG. 2 shows theresults of the experiment, demonstrating that immune complexes of CD154in complex with C4LB5, C4LB89, C4LB150, C4LB189, C4LB191 or C4LB199failed to activate platelets. CD154/C4LB94 IC induced PAC-1 onplatelets. The results demonstrate that anti-CD154 antibodies withIgG1sigma, IgG1sigmaYTE, IgG2sigma or IgG2sigmaYTE isotypes in generalmay not activate platelets, and may therefore have an improved safetyprofile over the antibodies on wild type IgG1.

Example 7. Effect of Isotype Switching on Antibody Properties

Variable regions of antibody C4LB89 were cloned as IgG1sigma/κ andIgG1sigmaYTE isotypes to assess possible differences in functionalityand developability. The new antibodies were named C4LB231 (IgG1sigma)and C4LB232 (IgG1sigmaYTE).

The resulting IgG1sigma and IgG1sigmaYTE antibodies were compared to theparental antibody in their functionality. C4LB231 and C4LB232 werecomparable in function to the parental C4LB89. Table 15 shows the IC₅₀values or a range of the IC₅₀ values for each antibody in the functionalassays as indicated in the Table. The IC₅₀ range in the table representsthe lowest and the highest IC₅₀ value obtained in experiments across 2-4donors with 1-6 repetitions. A singular IC₅₀ value in the tableindicates availability of one valid IC₅₀ value.

Table 15 CD154 format used to induce mAb Assay signaling C4LB231 C4LB232NF-κB SEAP reporter D1.1 Jurkat 0.27 gene assay; IC₅₀ (nM) NF-κB SEAPreporter shCD154-ILZ 1.21 1.25-1.45 gene assay; IC₅₀ (nM) NF-κB SEAPreporter shCD154-his 2.15 1.77-1.99 gene assay; IC₅₀ (nM) Jurkatcell-mediated D1.1 Jurkat 0.02-0.04 0.03-0.03 dendritic cell activationassay; IL-12p40 IC₅₀ (nM) human B cell D1.1 Jurkat 0.01-0.01 0.01-0.01proliferation IC₅₀ (nM) human B cell shCD154-ILZ 0.38-0.67 0.42-0.74proliferation IC₅₀ (nM) cyno B cell shCD154-ILZ 0.25-0.55 0.20-0.55proliferation IC₅₀ (nM)

C4LB231 and C4LB232 were also tested for their effect on platelets.Neither shCD154:C4LB231 nor shCD154:C4LB232 ICs activated platelets overthe baseline. CD154/5c8IgG1 IC activated platelets, and the activationwas blocked in the presence of IV.3, demonstrating that plateletactivation was mediated by IC binding to FcγRIIa. FIG. 3 shows theresults of the experiment.

Example 8. Anti-CD154 Antibodies Bind Human CD154 with High Affinity

Affinity measurements were done using ProteOn™ as described inExample 1. The on-rate, off-rate and affinity are shown in Table 16. Theparameters reported in this table were obtained from a 1:1 Langmuirbinding model for all samples, except C4LB94 and C4LB150 that fittedwith two-states binding model.

TABLE 16 Sample ka (1/Ms) kd (1/s) K_(D) (M) C4LB5 5.70E+05 1.78E−043.12E−10 C4LB89 1.61E+06 3.80E−04 2.35E−10 C4LB94 1.58E+06 3.29E−032.09E−09 C4LB150 2.27E+06 6.17E−03 2.72E−09 C4LB189 3.55E+05 2.06E−045.81E−10 C4LB191 5.62E+06 1.76E−04 3.13E−11 C4LB199 1.60E+06 4.04E−042.53E−10

Example 9. Crystal Structure of Marmoset CD154 in Complex with C4LB89

The epitope of antibody C4LB89 was identified using X-raycrystallography. The His-tagged Fab fragment of C4LB89 and theHis-tagged soluble form of marmoset CD40L (smCD154-his) were expressedin HEK293 GnTI cells and purified using affinity and size-exclusionchromatography. The smCD154:C4LB89 complex was incubated overnight at 4°C., concentrated, and separated from the uncomplexed species usingsize-exclusion chromatography. The complex was crystallized by thevapor-diffusion method from solution containing 16% PEG 3350, 0.2 Mammonium citrate, 0.1 MMES, pH 6.5. The crystals belong to the cubicspace group P2₁3 with unit cell dimensions of 162.1 Å. The structure ofthe complex was determined by the molecular replacement method using thecrystal structures of the C4LB89 Fab and CD40L (PDB entry 1ALY) assearch models.

The smCD154:C4LB89 complex is a symmetric trimer sitting on thecrystallographic 3-fold axis. C4LB89 binds mCD154 at the interfacebetween two subunits at the epitope distal from the cell surface. Theepitope includes 16 residues, 8 from each of two CD154 subunits. Theepitope residues are E182, 5185, Q186, A187, P188, 5214, A215 and R207in the first CD154 subunit and T176, F177, C178, Q220, S248, H249, G250and F253 in the second CD154 subunit. Epitope residue numbering isaccording to full length human CD154 of SEQ ID NO: 1. The paratope isdefined as antibody residues within 4 Å from the CD154 residues. TheC4LB89 paratope includes 9 residues from the heavy chain of C4LB89: S31and Y32 from HCDR1, S52, I54, F55 and N57 from HCDR2 and R100, Y101 andY102 from HCDR3. Paratope residue numbering is according to C4LB89 VH ofSEQ ID NO: 59. The light chain is not involved in contacts with mCD154.Based on the number of contacts, F55 in HCDR2 is a key antigenrecognition element. F55 makes contact to CD154 residues T176, F177,C178, Q220, S248, H249, G250 and F253. HCDR3 residues Y101 and Y102 alsocontribute to binding. FIG. 4 shows the HCDR2 and HCDR3 contact residuesand lack of binding of the LC to CD154. FIG. 5 shows a cartoon of theepitope and paratope residues.

Human and marmoset soluble CD154 proteins differ by 8 amino acidresidues only. All mCD154 C4LB89 epitope residues are conserved inhuman.Therefore, it is expected that the epitope is conserved between marmosetand human CD154. The alignment of the full length human and marmosetCD154 proteins are shown in FIG. 6.

Example 10. Platelet Activation by Anti-CD154 Antibodies is EpitopeDependent

C4LB89 variable regions (VH of SEQ ID NO: 59 and VL of SEQ ID NO: 66)were cloned as IgG1/κ, resulting in C4LB237 antibody. C4LB237 wasconfirmed to retain binding human CD154 (Table 17) as measured usingProteOn™ as described in Example 1. The affinity of C4LB237 to humanCD154 was 23.6±5.4 pM. Switching isotype of the VH/VL derived fromC4LB89 from IgG2sigma to IgG1 appeared to change the binding affinity ofthe resulting antibody.

As expected, C4LB237 bound human FcγRIIa and FcγRIIIa with a K_(D) of0.994 μM and 0.146 μM, respectively. C4LB237 showed comparable potencyto C4LB231 and higher potency than C4LB89 in NF-κB SEAP reporter geneassay when shCD154-his was used to induce signaling (Table 18).

C4LB237 was tested for its effect on platelet activation.shCD154:C4LB237 ICs did not activate platelets over the baseline, asshown in FIG. 7. This result suggests that in addition to the Fc, theepitope of the antibody contributes to the ability or inability of theantibody to activate platelets.

TABLE 17 ka (1/Ms) kd (1/s) Sample 10⁶ 10⁻⁰⁵ K_(D) (pM) C4LB231 (n = 8)2.53 ± 0.15 7.81 ± 0.69 31.0 ± 3.3 C4LB232 (n = 8) 2.54 ± 0.15 8.89 ±0.91 35.2 ± 5.2 C4LB237 (n = 4) 2.59 ± 0.20 6.05 ± 0.98 23.6 ± 5.4

TABLE 18 95% CI mAb IC₅₀ (nM) IC₅₀ (nM) C4LB231 2.32 2.11-2.55 C4LB2372.56 2.43-2.70 C4LB89 6.22 5.27-7.34

Example 11. Engineering Neutral Mutations on C4LB89

Analyses of the crystal structure of C4LB89 in complex with CD154revealed positions in the CDRs of C4LB89 which may be mutated withoutaffecting the overall structure of the complex and which therefore areexpected not to affect properties of the C4LB89 antibody. These neutralmutations on the light chain CDRs are listed in Table 19 and in theheavy chain CDRS in Table 20. The numbering of the residues that can bemutated is shown both on the individual CDRs and the VL or the VH. Forexample, residue Q4 on LCDR1 of SEQ ID NO: 37 (RASQSISSYLN) may bemutated to A, C, D, E, F, G, H, I, K, L, M, N, R, S, T, V, W or Y withthe expectation that the characteristics of the antibody do not changesubstantially. The corresponding residue in the VL of SEQ ID NO: 66 isQ27.

Mutations shown in Table 19 or Table 20 are made singularly or incombination onto C4LB89 using standard methods. The resulting VH/VLpairs are expressed and the mutated antibodies isolated andcharacterized using methods described herein.

TABLE 19 C4LB89 C4LB89 VL (SEQ LCDR ID NO: 66) C4LB89 LCDR residueresidue Possible substitutions LCDR1 SEQ ID Q4 Q27A, C, D, E, F, G, H, I, K, L, NO: 37 M, N, R, S, T, V, W, Y(RASQSISSYLN), S5 S28 A, C, D, E, F, G, H, I, K, L,M, N, Q, R, T, V, W, Y S7 S30 A, C, D, E, F, G, H, I, K, L,M, N, Q, R, T, V, W, Y S8 S31 A, C, D, E, F, G, H, I, K, L,M, N, Q, R, T, V, W, Y CDR2 of SEQ ID A2 A51 S NO: 44 N3 N52A, C, D, E, F, G, H, I, K, L, (YANSLQS) M, Q, R, S, T, V, W, Y S4 S53A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y L5 L54A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, Y Q6 Q55 E, D, N S7S56 A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W, Y LCDR3 of SEQ S3S91 A ID NO: 52 D4 D92 N (QQSDSIPWT), S5 S93A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V. W, Y I6 I94A, C, D, E, G, K, L, M, N, Q, R, S, T, V

TABLE 20 C4LB89 VH (SEQ C4LB89 ID NO: 59) HCDR Residue C4LB89 HCDRresidue position Possible substitutions HCDR1 SEQ ID S1 S31A, C, D, E, G, I, K, L, M, N, NO: 17 (SYGIS) Q, R, T, V I4 I34 M, L, VS5 S35 A HCDR2 of SEQ S3 S52 A, T, V ID NO: 23 I5 I54 V, T, L Q, E(WISPIFGNTNY N8 N57 A, C, D, E, F, G, H, I, K, L, AQKFQG)M, Q, R, S, T, V, W, Y T9 T58 A, C, D, E, F, G, H, I, K, L,M, N, Q, R, S, V. W, Y N10 N59 A, C, D, E, F, G, H, I, K, L,M, Q, R, S, T, V, W, Y HCDR3 of SEQ S1 S99 A, M ID NO: 30 R2 R100A, S, Q, K (SRYYGDLDY), R7 L105 M

Example 12. Evaluation of Platelet Activation and Higher Order ImmuneComplex Formation

Upon evaluation of the crystal structure data for Fab region and shCD154complex for C4LB231 and 5C8IgG1 earlier experiments with SC-HPLC and DLSas well as review of the platelet activation data, it was hypothesizedthat slight differences in binding of anti-CD154 antibodies with theshCD154 trimer may facilitate higher order immune complex formation: 1)the C4LB231 Fab binds between 2 subunits of the sCD154 trimer while 5C8Fab binds 1 subunit of sCD154, 2) the C4LB231 Fab appears to be morerigid in this conformation than the 5c8 Fab 3) and the angle of bindingof the anti-CD154 antibody with sCD154.

In order to further evaluate the role of the antibody epitope mediatingplatelet activation and higher order immune complex formation withshCD154, the VH and the VL of various anti-CD154 antibodies were clonedand expressed as Fc silent IgG2sigma and IgG1sigma isotypes or as IgG1.The generated antibodies are shown in Table 21. Platelet activationassays were done as described in Example 6. For the higher order immunecomplex formation evaluations via size exclusion high performance liquidchromatography (SE-HPLC) and dynamic light scattering (DLS), theantibodies were complexed with shCD154 at 1:1 (aka 10:10) and 10:1 molarratios to evaluate if there were concentration dependent differences inimmune complexation.

TABLE 21 Antibody VH SEQ ID VL SEQ ID name Isotype NO: NO: 5C8IgG2sigmaIgG2σ 74 75 (C4LB71) 5C8IgG1 IgG1 (MSCB8) C4LB89 IgG2σ 59 66 C4LB231IgG1 C4LB237 IgG1 C4LB119 IgG2σ 84 85 C4LB290 IgG1 C4LB287 IgG1 C4LB83IgG2σ 86 87 C4LB288 IgG1 C4LB94 IgG2σ 60 67 C4LB234 IgG1σ C4LB289 IgG1IgG2σ: IgG2sigma IgG1σ: IgG1sigma

VH of C4LB119 SEQ ID NO: 84QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYYISWVRQAPGQGLEWMGAIDPYFGYANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARTG LNYGGFDYWGQGTLVTVSSVL of C4LB119 SEQ ID NO: 85EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQ GTKVEIK VH of C4LB83SEQ ID NO: 86 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGWIIPIFGNTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREK DFRGYTKLDYWGQGTLVTVSSVL of C4LB83 SEQ ID NO: 87DIQMTQSPSSLSASVGDRVTITCRASQSINNWLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSFPYTFGQ GTKVEIK

C4LB83 and C4LB119 binding characteristics and functionality wasassessed using protocols described in Example 1 and are shown in Table22.

TABLE 22 B cell activation assay IC₅₀ Antibody ka (1/Ms) kd (1/s) K_(D)(M) (nM)* C4LB83 2.10E+06 4.70E−03 2.23E−09 0.64-1.09 C4LB119 1.84E+068.80E−03 4.79E−09 0.81-2.03 *shCD154-LZ induced activation of human Bcells. The range indicates the lowest and highest IC₅₀ value obtained inseparate experiments across 2 donorsMethodsSE-HPLC

Immune complexes of antibodies shown in Table 21 and Alexafluo-448labeled shCD154 were prepared at 1:1 and 10:1 antibody:shCD154 molarratio in 110 μL of 1×PBS and incubated at 37° C. for 30 min. 100 μL ofeach sample were injected to column (Agilent, 1100/1200 system) for eachrun, which contained 5 μg of AF488 labeled shCD154 and 15 μg (1:1ratio), or 150 g (10:1 ratio) of anti-CD154 mAb. Molecular weight wascalculated based on the retention time of the standards (Bio-Rad). Therelationship between the molecular weight and retention time of theprotein satisfies the equation:log(M)=b−cT,where M is the molecular weight, T is the retention time, and b and care constants. A linear curve of log(M) and T was obtained from thestandard SE-HPLC chromatogram by least squares fitting, with R²=0.9928.DLS

DLS measurement is based on light scattering principles and molecular orBrownian Motion. Brownian motion, a typically behavior of molecules insolution, causes light scattered to be both in and out of phase,resulting in constructive and destructive interferences. The result isthat the intensity of the scattered light fluctuates with time. DuringDLS or QELS (quasi-electric light scattering) analysis, thistime-dependent fluctuation in the scattered light intensity is capturedby a fast photon counter. The fluctuations are directly proportional tothe rate of diffusion. Resolution of the correlation data using theStokes-Einstein equation yields the hydrodynamic radius. UnlikeSEC-MALS, or SEC, where sample resolution can distinguish betweenmonomer and dimer, DLS is only able to resolve species that areseparated by a size factor of ˜4, therefore monomer and tetramer willbegin to be resolved; however monomer through dimer will not, but willreport a weighted average of the mixture.R _(h) =kT/6πηD;  Stokes-Einstein equation:

wherein

D=diffusion coefficient,

k=Boltzmann's constant

T=temperature

η=viscosity

Antibodies alone at 10 μM or immune complexes of antibodies shown inTable 21 and shCD154 were prepared at 10:1 or 10:10 molar concentrationsof antibody:shCD154 (10 μM antibody with either 1 μM or 10 μM shCD154)in PBS. All samples were prepared in glass vials with vial inserts (250μL deactivated glass with polymer feet, Agilent cat #5181-8872), andsamples were added in the following order: PBS, mAb, shCD154. Sampleswere mixed by gentle vortexing then incubated at RT, with rocking(Nutating Mixer (VWR) ˜30 rev/min for approximately 23 hr. If needed,antibodies were first concentrated using standard methods.

Particle sizes and species distribution in all samples were determinedon a DynaPro® Plate Reader DLS instrument (Wyatt TechnologiesCorporation) at 23° C. DLS consistency was first validated using BSA(data not shown). Measurements were made by introducing 30 μL of samplein each of 3 wells for (triplicate measurements). DLS measurements wereacquired in a DynaPro® plate reader. Twenty 5-sec acquisitions wereperformed on each sample, and laser power was automatically adjusted bythe instrument. Parameters used for data analysis included a viscosityat 23° C. for PBS of 1.019 cP and a refractive index value at 589 nm &23° C. for PBS of 1.333. A globular protein model was used by theprogram. Signals were bucketed into peaks, where Peak1 was 0.1-10 nm,Peak2 was 10-100 nm, Peak3 was 100-1000 nm and Peak4 was 1000-5000 nm.Visible precipitates (if formed) in the sample were noted. Data analysiswas performed using Dynamics software (Wyatt Technology Inc.). Plots ofpercent mass versus species radius (Rh) were generated. Peak radius,polydispersity, percent mass and percent intensity were calculated andrecorded.

Results

Platelet Activation

Ability of the immune complexes of shCD154 and antibodies expressed asFc silent IgG2sigma or IgG1sigma, or as wild-type IgG1 to activateplatelets were assessed. 5c8 antibody cloned on various IgG scaffoldswas used as a control. FIG. 3 shows that 5c8IgG1:shCD154 immunecomplexes activated platelets in FcγRIIa-dependent manner, as ananti-FcγRIIa antibody inhibited 5c8IgG1-mediated platelet activation.5c8 VH/VL regions cloned on effector silent Fc, IgG1sigma or IgG2sigmalost their ability to activate platelets (FIG. 1). These results areconsistent to what has been described earlier. However, unexpectedly,experiments conducted in Example 10 demonstrated that immune complexeswith wild-type IgG1 antibody (C4LBB237:shCD154) did not activateplatelets (FIG. 7), prompting further studies into possible epitopedependency of platelet activation.

The VH/VL regions of 4 distinct antibodies were cloned as IgG2sigma,IgG1sigma or wild-type IgG1 in order to evaluate the effect of theepitope and/or Fc on platelet activation as well as on formation ofhigher order immune complexes. Contrary to what has been disclosedearlier in literature, it was found that platelet activation was in somecases mediated by the antibody epitope independent on FcγRIIa-mediatedcross-linking.

FIG. 8A, FIG. 8B and FIG. 8C shows the summary of the analyses. Immunecomplexes of Fc silent antibodies C4LB119 (FIG. 8A) and C4LB94 (FIG. 8B)activated platelets in FcγRIIa-independent manner, as pre-block with ananti-FcγRIIa antibody failed to inhibit platelets activation. IC ofC4LB83, also an Fc silent antibody, moderately activated platelets (FIG.8C). IC of antibodies with identical VH/VL domains cloned on wild-typeIgG1 in each case activated platelets in FcγRIIa-mediated fashion(C4LB278 in FIG. 8A, C4LB289 in FIG. 8B, and C4LB288 in FIG. 8C).Therefore, several antibodies were identified which mediated plateletactivation despite of having a silent Fc. One VH/VL domain pair wasidentified which mediated platelet activation neither on silent(C4LB231) nor wild-type IgG1 (C4LB237). These data demonstrate thatantibody epitope plays a role in mediating platelet aggregation.

Higher Order Immune Complex Formation

SE-HPLC and DLS were used to further evaluate the presence of higherorder immune complex formation and the approximate size of these immunecomplexes between anti-CD154 antibodies shown in Table 21 and shCD154.As shCD154 is a trimer in solution, the expected stoichiometry of theantibody:shCD154 trimer in solution is 3:1. For SE-HPLC, heavier andthus larger immune complexes would have shorter retention times, whilelighter and thus smaller immune complexes would have longer retentiontimes, with the exception of very large immune complexes that are unableto elute off the column and evidenced by low % recovery. For DLS, thelarger the radius (Rh) value the larger the immune complex. Plate DLStechniques cannot resolve IgG monomers, dimers, trimers and tetramers.Thus Rh values obtained will be a weighted average of monomer—tetramer,therefore Rh values that are closer to 6.5 could represent solutions ofmAbs containing higher ordered species, typically dimer. Where bindingof mAb to shCD154 is stoichiometric two species may be present—the 3:1complex (˜500 kDa) and unbound antibody (˜150 kDa) which corresponds toRh values of ˜8.8 nm and 5-6.5 nm for the 3:1 complex and free mAbrespectively. Neither technique can exactly resolve the molecular weightof the immune complexes, but can allow comparative sizes. Table 23 showsthe relationship of retention time and hydrodynamic radii to approximatemolecular weights.

TABLE 23 Approximate SE-HPLC DLS Molecular Retention Hydrodynamic Weighttime Radius Immune Complex (kDa) (min) (nm) mAb 150 ~9 min 5 to 6.5mAb:shCD154 550 ~7 min ~8.8 (trimer) Higher order immune ~1000 <7 min~12 complexesDLS

mAbs in the absence of CD154 and antibody:shCD154 immune complex sizeswere evaluated in conditions in which antibody:shCD154 complexes wereformed in excess of antibody (10:1 molar ratio) and at equivalentconcentration (1:1 molar ratio). Under the conditions where antibody isin excess, the antibody typically saturates CD154 sites to form theimmune complex, and excess unbound antibody is present. Under equivalentconcentration, no free antibody or free shCD154 is present. The Rhvalues obtained for each mAb in the absence of sh CD154 and in thepresence of sh CD154 at 10:1 and 10:10 molar ratios are shown in Table24.

A typical mAb with a nominal MW of 150 kDa yields an Rh value of 5.0-6.5nm. IgG dimers, often seen during size exclusion chromatography, cannotbe resolved by plate DLS techniques. The Rh values obtained will be aweighted average of monomer—tetramer, therefore Rh values that arecloser to 6.5 could represent solutions of mAbs containing higherordered species, typically dimer.

For mAbs in the absence of CD154, expected R_(h) values of 5.5-6.3 nmwere observed for all mAbs except C4LB287 and C4LB234, with R_(h) valuesof 6.9, and 7.1 nm, respectively, suggested that these antibodies haveinherent aggregation tendencies.

The Rh values for the antibody:shCD154 complexes formed at 10:1 ratiowere generally less than about 8.8 nm, indicative of the 3:1stochiometric complex without formation of higher order immunecomplexes, except for 5c8IgG2sigma (C4LB71) and C4LB234 (R_(h) values8.9 and 9.3 nm respectively), indicating that higher order immunecomplexes were formed.

Increasing shCD154 concentration to 10 μM resulted in antibody:shCD154immune complexes with elevated Rh when compared to the complexes with 1μM CD154 (10:1 antibody:CD154 ratio). Some of the antibody:shCD154complexes demonstrated heterogeneicity with high molecular weightsecondary species present ranging from of the total mass. C4LB71,5C8IgG1, C4LB89, C4LB119, C4LB94, C4LB234 and C4LB289 IC Rh values were22.1, 615, 56.3, 45.0, 18.3, 18.7 and 19.6 nm respectively. In addition,C4LB89, C4LB119, C4LB83, C4LB228 also formed species 900-4000 nm, andC4LB89 and C4LB119 formed precipitates indicating very large immunecomplexes. Species near 12 nm Rh typically correspond to a mass of˜1,000 kDa, therefore these mAbs form very large immune complexes withCD154 under these conditions.

TABLE 24 Hydrodynamic radius (Rh), nm Antibody Isotype 10:0* 10:1*10:10* 5c8IgG2a IgG2σ 5.8 8.9 22.1 (C4LB71) 5c8IgG1 IgG1 5.7 8.3 19.6C4LB89 IgG2σ 5.9 5.3, (4082)  615 ppt C4LB231 IgG1σ 5.9 8.2 10.5 C4LB237IgG1 6.1 7.4 9.8 C4LB119 IgG2σ 6.3 8.0 56.3 ppt C4LB290 IgG1σ 6.1 8.313.3 C4LB287 IgG1 6.9 8.2 12.9 C4LB83 IgG2σ 6 6.1 14.5, (3632)  C4LB288IgG1 6.1 6.6 10.9 C4LB94 IgG2σ 6.1 6.9 45 C4LB234 IgG1σ 7.1 9.3, (83)5.3, (18.3) C4LB289 IgG1 5.6, 7.8, (17, 1350) 3.7, (18.7)*Antibody:shCD154 ratio Rh of secondary species were included inparenthesis if the % mass was ≥25% ppt: solution precipitated IgG2σ:IgG2sigma IgG1σ: IgG1sigmaSE-HPLC

Table 25 shows the Retention time, recovery rate, and estimatedmolecular weight (MW) of anti-CD154 antibodies alone and in immunecomplex with shCD154 obtained from the SE-HPLC analyses. Atypicalantibody has a MW of about 150 kD, and shCD154 trimer has a MW of about50 kDa. Therefore, a mAb:shCD154 trimer complex with 3:1 stoichiometryhas an expected MW of about 500 kDa.

TABLE 25 Retention Time Peak % MW mAb ID Type Ratio* (min) Area Recovery(kDa) 5c8IgG2σ IgG2σ 1:1 6.24 6887 85.2 1596.8 (C4LB71) 10:1  6.28 734890.9 1538.2 5c8IgG1 IgG1 1:1 7.3 1096 55.7 592.8 10:1  6.92 3280 69.2845.6 C4LB89 IgG2σ 1:1 6.19 356 16.9** 1673.2 10:1  6.35 3662 45.3**1440.8 C4LB231 IgG1σ 1:1 6.97 6766 83.8 807 10:1  6.97 7181 88.9 807C4LB237 IgG1 1:1 6.99 6506 80.5 792.1 10:1  6.99 7977 98.7 792.1 C4LB119IgG2σ 1:1 9.59 307 5.6** 69.7 10:1  8.02 4667 8.3** 302.4 C4LB290 IgG1σ1:1 7.01 4957 61.4 777.4 10:1  7.01 6983 86.4 777.4 C4LB287 IgG1 1:1 75937 73.5 784.7 10:1  7.02 8011 99.2 770.2 C4LB83 IgG2σ 1:1 7.86 519364.3 351.2 10:1  7.84 3355 86.9 357.8 C4LB288 IgG1 1:1 7.62 4169 70.6439.5 10:1  7.37 5582 95.5 555.2 C4LB94 IgG2σ 1:1 6.01 3233 40.0**1979.9 10:1  6.02 7748 95.9 1961.5 C4LB234 IgG1σ 1:1 6.26 5534 86.61567.3 10:1  6.53 7922 98.1 1217.6 C4LB289 IgG1 1:1 6.25 5800 83.5 158210:1  6.53 7958 98.5 1217.6 *(mAb:CD154) **Recovery rate <50% Antibodieswith identical VH/VL are separated into groups divided by empty rowsIgG2σ: IgG2sigma IgG1σ: IgG1sigma

All antibodies formed an immune complex with shCD154. C4LB231, C4LB237,C4LB290, C4LB287 (all IgG1 sigma) formed an immune complex with sCD154and eluted at 7.0 min however C4LB290 and C4LB287 had lower % recoveriesat the 1:1 condition compared to the 10:1 condition while C4LB231 andC4LB237 had higher % recoveries (>80%). C4LB289 (IgG1), C4LB234(IgG1sigma), 5c8IgG1sigma (C4LB71) and C4LBB94 (IgG2sigma) elutedearlier at 6.2 to 6.5 min indicating larger complexes were formed thanwith the aforementioned mAbs. 5c8IgG1 had abroad peak at the expectedretention time for the 3:1 mAb:shCD154 trimer immune complex, howeverthe broad peak and lower recovery (56% at the 1:1 condition) indicatedthat higher order immune complexes may have formed which may have eitherinteracted with or did not enter the column. C4LB89 and C4LB119 (bothIgG2sigma) formed complex with shCD154 and eluted with a broad peak andvery low recovery (6-17% at the 1:1 condition), likely due to theformation of large complexes which did not enter the column. In general,antibodies on IgG2sigma isotypes formed larger immune complexes whencompared to the antibodies on IgG1sigma or IgG1.

Table 26 shows a summary of antibody characteristics. Overall, theplatelet activation data, the SE-HPLC and DLS data together indicatethat platelet activation is not entirely attributed to an active Fc. Thedata indicates that antibodies with silent Fc such as IgG1sigma andIgG2sigma are capable of forming larger size immune complexes greaterthan that of the expected 3:1 mAb to shCD154 trimer complex and thatsome antibodies on silent Fc are capable of platelet activation. Thedata supports a conclusion that both the VH/VL domains (e.g. the epitopethe antibody binds to) and the higher order immune complex formationcontribute to platelet activation.

TABLE 26 Platelet activation IC SE-HPLC Platelet independent RT (min) ICDLS Rh (nm) Antibody Isotype activation** on FcγRIIa 10:1* 1:1* 10:1*10:10* 5c8IgGσ IgG2σ No 6.3 6.2 8.9 22.1 (C4LB71) 5c8IgG1σ IgG1σ No5c8IgG1 IgG1 Yes No 6.9 7.3 8.3 19.6 C4LB89 IgG2σ No 6.4^(#) 6.2^(#) 5.3, 615 p  4082{circumflex over ( )}   C4LB231 IgG1σ No 7.0 7.0 8.210.5 C4LB237 IgG1 No 7.0 7.0 7.4  9.8 C4LB119 IgG2σ Yes Yes 8.0^(#)9.6^(#) 8.0   56.3 p C4LB290 IgG1σ 7.0 7.0 8.3 13.3 C4LB287 IgG1 Yes No7.0 7.0 8.2 12.9 C4LB83 IgG2σ Marginal Yes 7.8 7.9 6.1  14.5,3632{circumflex over ( )}   C4LB288 IgG1 Yes No 7.4 7.6 6.6 10.9 C4LB94IgG2σ Yes Yes 6.0 6.0^(#) 6.9 45   C4LB234 IgG1σ 6.5 6.3 9.3   5.3,  18.3{circumflex over ( )} C4LB289 IgG1 Yes No 6.5 6.3 7.8   3.7, 18.7*antibody:shCD154 ratio **assessment using either PAC-1 or CD62pexpression ^(#)SE-HPLC % recovery <50% {circumflex over ( )}Rh ofsecondary species were included if the % mass was ≥25% p: solutionprecipitated IgG2σ: IgG2sigma IgG1σ: IgG1sigma

It is claimed:
 1. A polynucleotide encoding an antagonistic antibody oran antigen binding portion thereof specifically binding human CD154 ofSEQ ID NO: 1, comprising a light chain complementarity determiningregion (LCDR) 1 of SEQ ID NO: 37 (RASQSISSYLN), a LCDR2 of SEQ ID NO: 44(YANSLQS), a LCDR3 of SEQ ID NO: 52 (QQSDSIPWT), a heavy chaincomplementarity determining region (HCDR) 1 of SEQ ID NO: 17 (SYGIS), aHCDR2 of SEQ ID NO: 23 (WISPIFGNTNYAQKFQG), and a HCDR3 of SEQ ID NO: 30(SRYYGDLDY), wherein optionally: a) the LCDR1 residue Q4 is mutated toA, C, D, E, F, G, H, I, K, L, M, N, R, S, T, V, W or Y; b) the LCDR1residue S5 is mutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V,W or Y; c) the LCDR1 residue S7 is mutated to A, C, D, E, F, G, H, I, K,L, M, N, Q, R, T, V, W or Y; d) the LCDR1 residue S8 is mutated to A, C,D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W or Y; e) the LCDR2 residueA2 is mutated to S; f) the LCDR2 residue N3 is mutated to A, C, D, E, F,G, H, I, K, L, M, Q, R, S, T, V, W or Y; g) the LCDR2 residue S4 ismutated to A, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W or Y; h)the LCDR2 residue L5 is mutated to A, C, D, E, F, G, H, I, K, M, N, Q,R, S, T, V, W or Y; i) the LCDR2 residue Q6 is mutated to E, D or N; j)the LCDR2 residue S7 is mutated to A, C, D, E, F, G, H, I, K, L, M, N,Q, R, T, V, W or Y; k) the LCDR3 residue S3 is mutated to A; l) theLCDR3 residue D4 is mutated to N; m) the LCDR3 residue S5 is mutated toA, C, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W or Y; n) the LCDR3residue I6 is mutated to A, C, D, E, G, K, L, M, N, Q, R, S, T or V; o)the HCDR1 residue S1 is mutated to A, C, D, E, G, I, K, L, M, N, Q, R, Tor V; p) the HCDR1 residue I4 is mutated to M, L or V; q) the HCDR1residue S5 is mutated to A; r) the HCDR2 residue S3 is mutated to A, Tor V; s) the HCDR2 residue P4 is mutated to V, T, L Q or E; t) the HCDR2residue N8 is mutated to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V,W or Y; u) the HCDR2 residue T9 is mutated to A, C, D, E, F, G, H, I, K,L, M, Q, R, S, T, V, W or Y; v) the HCDR2 residue N10 is mutated to A,C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y; w) the HCDR3residue S1 is mutated to A or M; x) the HCDR3 residue R2 is mutated toA, S, Q or K; or y) the HCDR3 residue L7 is mutated to M.
 2. Thepolynucleotide of claim 1, wherein the antibody or antigen bindingportion thereof comprises the HCDR1 of SEQ ID NO: 17 (SYGIS), the HCDR2of SEQ ID NO: 23 (WISPIFGNTNYAQKFQG), the HCDR3 of SEQ ID NO: 30(SRYYGDLDY), the LCDR1 of SEQ ID NO: 37 (RASQSISSYLN), the LCDR2 of SEQID NO: 44 (YANSLQS) and the LCDR3 of SEQ ID NO: 52 (QQSDSIPWT).
 3. Thepolynucleotide of claim 1, wherein the antibody has at least one of thefollowing properties: a) an immune complex of the antibody and solublehuman CD154 (shCD154) does not activate platelets, wherein plateletactivation is measured by P-selectin surface expression on platelets; b)binds to CD154 with a dissociation constant (K_(D)) of about 5×10⁻⁹ M orless, when the K_(D) is measured using ProteOn XPR36 system at 25° C. inDulbecco's phosphate buffered saline containing 0.03% polysorbate P20and 100 μg/ml bovine serum albumin; c) inhibits CD154-mediated human Bcell proliferation with an IC₅₀ value of about 2.7×10⁻⁹ M or less; or d)inhibits CD154-mediated expression of secreted embryonic alkalinephosphatase (SEAP) under NF-κB-inducible interferon-β (IFN-β) minimalpromoter in HEK293 cells stably expressing SEAP and human CD40 with anIC₅₀ value of about 2.1×10⁻⁸M or less.
 4. The polynucleotide of claim 1,wherein the human CD154 is a homotrimer and the antibody binds a firstCD154 monomer in the homotrimer within amino acid residues 182-207 ofCD154 and a second CD154 monomer in the homotrimer within amino acidresidues 176-253 of CD154, wherein residue numbering is according to SEQID NO:
 1. 5. The polynucleotide of claim 4, wherein the antibody bindsresidues E182, S185, Q186, A187, P188, S214, A215 and R207 in the firstCD154 monomer, wherein residue numbering is according to SEQ ID NO: 1.6. The polynucleotide of claim 4, wherein the antibody binds residuesT176, F177, C178, Q220, S248, H249, G250 and F253 in the second CD154monomer, wherein residue numbering is according to SEQ ID NO:
 1. 7. Thepolynucleotide of claim 1, wherein the antibody is of IgG1, IgG2, IgG3or IgG4 isotype.
 8. The polynucleotide of claim 7, wherein the antibodyor antigen binding portion thereof comprises at least one substitutionin an Fc region, wherein the at least one substitution in the Fc regionis a substitution L234A, L235A, G237A, P238S, M252Y, S254T, T256E,H268A, A330S or P331S, wherein residue numbering is according to the EUIndex.
 9. The polynucleotide of claim 8, wherein the antibody or antigenbinding portion thereof comprises substitutions L234A, L235A, G237A,P238S, H268A, A330S or P331S in the Fc region, wherein residue numberingis according to the EU Index.
 10. The polynucleotide of claim 7, whereinthe antibody or antigen binding portion thereof comprises at least onesubstitution in an Fc region, wherein the at least one substitution inthe Fc region is a substitution V234A, G237A, P238S, M252Y, S254T,T256E, H268A, V309L, A330S or P331S, wherein residue numbering isaccording to the EU Index.
 11. The polynucleotide of claim 10, whereinthe antibody or antigen binding portion thereof comprises substitutionsV234A, G237A, P238S, H268A, V309L, A330S and P331S in the Fc region,wherein residue numbering is according to the EU Index.
 12. Thepolynucleotide of claim 7, wherein the antibody comprises a heavy chain(HC) and a light chain (LC) of SEQ ID NOs: a) 80 and 81, respectively;b) 82 and 81, respectively; or c) 83 and 81, respectively.
 13. Thepolynucleotide of claim 1, wherein the antibody or antigen bindingportion thereof is multispecific.
 14. The isolated polynucleotide ofclaim 1, wherein the human CD154 is a homotrimer and the antibody bindsa first CD154 monomer in the homotrimer within amino acid residues182-207 of CD154 and a second CD154 monomer in the homotrimer withinamino acid residues 176-253 of CD154, wherein residue numbering isaccording to SEQ ID NO:
 1. 15. A polynucleotide encoding an antagonisticantibody or an antigen binding portion thereof specifically bindinghuman CD154 of SEQ ID NO: 1, comprising a heavy chain complementaritydetermining region (HCDR) 1 of SEQ ID NO: 17 (SYGIS), a HCDR2 of SEQ IDNO: 23 (WISPIFGNTNYAQKFQG) and a HCDR3 of SEQ ID NO: 30 (SRYYGDLDY),wherein optionally a) the HCDR1 residue S1 is mutated to A, C, D, E, G,I, K, L, M, N, Q, R, T or V; b) the HCDR1 residue I4 is mutated to M, Lor V; c) the HCDR1 residue S5 is mutated to A; d) the HCDR2 residue S3is mutated to A, T or V; e) the HCDR2 residue P4 is mutated to V, T, L Qor E; f) the HCDR2 residue N8 is mutated to A, C, D, E, F, G, H, I, K,L, M, Q, R, S, T, V, W or Y; g) the HCDR2 residue T9 is mutated to A, C,D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W or Y; h) the HCDR2 residueN10 is mutated to A, C, D, E, F, G, H, I, K, L, M, Q, R, S, T, V, W orY; i) the HCDR3 residue S1 is mutated to A or M; j) the HCDR3 residue R2is mutated to A, S, Q or K; k) the HCDR3 residue L7 is mutated to M; anda light chain complementarity determining region (LCDR) 1, a LCDR2 and aLCDR3 of: a) SEQ ID NOs: 36, 43 and 51, respectively; b) SEQ ID NOs: 37,44 and 52, respectively; c) SEQ ID NOs: 38, 45 and 53, respectively; d)SEQ ID NOs: 39, 46 and 54, respectively; e) SEQ ID NOs: 40, 47 and 55,respectively; f) SEQ ID NOs: 41, 47 and 56, respectively; g) SEQ ID NOs:42, 48 and 57, respectively; h) SEQ ID NOs: 37, 49 and 52, respectively;or i) SEQ ID NOs: 37, 50 and 52, respectively.
 16. The polynucleotide ofclaim 15, the antibody or antigen binding portion thereof comprises aheavy chain variable region (VH) of SEQ ID NO:
 59. 17. Thepolynucleotide of claim 16, wherein the antibody or antigen bindingportion thereof comprises a light chain variable region (VL) of SEQ IDNOs: 65, 66, 67, 68, 69, 70, 71, 72 or
 73. 18. The polynucleotide ofclaim 17, wherein the antibody or antigen binding portion thereofcomprises the VL of SEQ ID NOs: 66, 72 or
 73. 19. The polynucleotide ofclaim 18, wherein the antibody or antigen binding portion thereofcomprises the VH of SEQ ID NO: 59 and the VL of SEQ ID NO:
 66. 20. Thepolynucleotide of claim 18, wherein antibody or antigen binding portionthereof comprises the VH of SEQ ID NO: 59 and the VL of SEQ ID NO: 72.21. The polynucleotide of claim 18, wherein antibody comprises the VH ofSEQ ID NO: 59 and the VL of SEQ ID NO:
 73. 22. An isolatedpolynucleotide encoding an antagonistic antibody or an antigen bindingportion thereof specifically binding CD154 of SEQ ID NO: 1, comprisinga) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3of SEQ ID NOs: 17, 23, 30, 37, 44 and 52, respectively; b) the VH of SEQID NO: 59 and the VL of SEQ ID NO: 66; or c) the heavy chain of SEQ IDNO: 80 and the light chain of SEQ ID NO:
 81. 23. The polynucleotide ofclaim 15, comprising the polynucleotide sequence of SEQ ID NOs: 76 or77.
 24. A vector comprising the polynucleotide of claim
 15. 25. A hostcell comprising the vector of claim 24.